Highly fluorinated oils and surfactants and methods of making and using same

ABSTRACT

Disclosed are compounds comprising the structure: 
                         
In one aspect, the compounds exhibit maximum symmetric branching. Also disclosed are bilayers, micelles, coatings, and nanoparticles comprising the disclosed compounds. Also disclosed are processes for the preparation of the disclosed compounds and methods of using the disclosed compounds. Also disclosed are highly fluorinated dendrons and methods for making same. Also disclosed are methods for Fluorous Mixture Synthesis and tagging. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 60/785,463,filed Mar. 24, 2006, which is hereby incorporated herein by reference inits entirety.

ACKNOWLEDGEMENT

This invention was made with government support under Grants Nos. EB004416 and EB 002880 awarded by the National Institutes of Health. TheUnited States government has certain rights in the invention.

BACKGROUND A. Highly Fluorinated Compounds

It is well known to employ fluorine groups in organic molecules toenhance the hydrophobicity of the compounds. Also, fluorinatedsurfactants generally have higher surface activities in comparison withtheir non-fluorinated counterparts. However, common fluorinatedcompounds, for example polytetrafluoroethylene, typically use onlyunbranched or asymmetrical fluorinated moieties. The consequence ofless-than-maximum branching is that the surface area of the fluorocarbonmoiety is not maximized. For surfactants, this is a distinct advantage.The consequence of asymmetrical branching is that the terminal groups ofthe fluorocarbon moiety have different chemical environment. This canalso reduce the effectiveness of surface protection. Also, for ¹⁹F MRIapplications, variation in chemical environments of fluorine atoms leadsto split ¹⁹F signals, thereby reducing the sensitivity for MRIdetection.

B. Targeted Radiotherapy

Targeted radiotherapy is a promising cancer treatment modality. Larson,S. M. & Krenning, E. P. A pragmatic perspective on molecularradionuclide therapy. [J. Nucl. Med. 46 (Suppl. 1) 1S-3S, 2005]. Intargeted radiotherapy, ionizing radionuclides (e.g., ⁹⁰Y, ¹³¹I) aredelivered to the tumor site via a targeting moiety to which they areattached. The targeting moiety can be monoclonal antibodies (mAb),peptides or any other molecules which recognize receptor molecules thatare over-expressed by tumor cells. Compared with external beamradiation, targeted radiotherapy has two significant advantages: it canreduce radiation damage to normal tissues and it can reach multiplesmall metastases. [de Jong, M., Kwekkeboom, D., Valkema, R. & Krenning,E. P. Radiolabelled peptides for tumour therapy: current status andfuture directions. Eur. J. Nucl. Med. & Mol. Imag. 463-469, 2003;Kaltsas, G. A., Papadogias, D., Makras, P. & Grossman, A. B. Treatmentof advanced neuroendocrine tumours with radiolabelled somatostatinanalogues. Endocrine-Related Cancer, 12, 683-699, 2005].

The emergence of targeted radiotherapy as an effective cancer treatmentmodality is demonstrated by the regulatory approvals of tworadioimmunotherapy drugs (Zevalin® and Bexxar®) for treatingnon-Hodgkin's lymphoma [Drug Label Information for Zevalin® and Bexxar®.Available at Drugs@FDA.]. In addition, OctreoTher®, which uses anoctapeptide for tumor targeting, is under clinical trials for treatingneuroendocrine tumors [Kaltsas, G. A., Papadogias, D., Makras, P. &Grossman, A. B., Treatment of advanced neuroendocrine tumours withradiolabelled somatostatin analogues. Endocrine-Related Cancer, 12,683-699, 2005; Smith, M. C., Liu, J., Chen, T., Schran, H., Yeh, C.-M.,Jamar, F., Valkema, R., Bakker, W., Kvols, L., Krenning, E. & Pauwels,S. OctreoTher™: ongoing early clinical development of asomatostatin-receptor-targeted radionuclide antineoplastic therapy;Bushnell, et al., Evaluating the clinical effectiveness of 90Y-SMT 487in patients with neuroendocrine tumors. J. Nucl. Med. 44, 1556-1560,2003].

The first step in developing targeted radiotherapeutic drugs is, ofcourse, drug discovery, which includes the identification of the target(i.e., which receptor) and the drug (which ligand and whichradio-nuclide). However, once a radiotherapeutic drug has beendiscovered, it still faces enormous challenges in its delivery, asevidenced by the complex dosing schedules of Zevalin® and Bexxar®, whichtake about two weeks [Drug Label Information for Zevalin® and Bexxar®.Available at Drugs@FDA.]. The origin of this delivery challenge lies inthe large variations among patients in terms of drug pharmacokineticsand tumor microenvironment. Pharmacokinetics refers to the distribution,retention and excretion profiles of the radiotherapeutic drug. Largepharmacokinetic variations among patients have been observed for bothmAb- and peptide-based radiopharmaceuticals [Kaltsas, G. A., Papadogias,D., Makras, P. & Grossman, A. B. Treatment of advanced neuroendocrinetumours with radiolabelled somatostatin analogues. Endocrine-RelatedCancer, 12, 683-699, 2005; Forsell-Aronsson, E., Bernhardt, P., Nilsson,O., Tissel, L.-E., Wangberg, B. & Ahlman, H. Biodistribtuin data from100 patients i.v. injected with 111In-DTPA-D-Phe-octreotide. ActaOncologica 43, 436-442, 2004; Barone, et al., Patient-specific dosimetryin predicting renal toxicity with 90Y-DOTATOC: relevance of kidneyvolume and dose rate in finding a dose-effect relationship. J. Nucl.Med. 99S-106S, 2005; Wahl, R. L. Tositumomab and 131I therapy innon-Hodgkin's lymphoma. J. Nucl. Med. 46 (Suppl. 1), 128S-140S, 2005].Tumor microenvironment refers to the physiological and metabolicconditions of tumors which also vary significantly from individual toindividual [Gillies, R. J., Raghunand, N., Karczmar, G. S. & Bhujwalla,Z. M. MRI of the tumor microenvironment. J. Mag. Reson. Imag. 16,430-450, 2002]. In the context of radiotherapy, the most relevant tumormicroenvironmental parameter is oxygen tension (pO₂). This is becausehypoxic tumor cells are more radioresistant [Vaupel, P. Tumormicroenvironmental physiology and its implications for radiationoncology. Seminars in Radiation Oncology, 14, 198-206, 2004] and pO₂ hasbeen found to correlate with treatment outcomes of beam radiation andtargeted radiotherapy [Nordsmark, M., Overgaard, M. & Overgaard, J.Pretreatment oxygenation predicts radiation response in advancedsquamous cell carcinoma of the head and neck. Radiother. Oncology, 41,31-39, 1996; Nordsmark, M. & Overgaard, J. A confirmatory prognosticstudy on oxygenation status and loco-regional control in advanced headand neck squamous cell carcinoma treated by radiation therapy.Radiother. Oncol. 57, 39-43, 2000; Fyles, et al., Oxygenation predictsradiation response and survival in patients with cervix cancer.Radiother. Oncology, 48, 149-156, 1998; Brizel, D. M., Dodge, R. K.,Clough, R. W. & Dewhirst, M. W. Oxygenation of head and neck cancer:changes during radiotherapy and impact on treatment outcome. Radiother.Oncology, 53, 113-117, 1999; O'Hara, J. A., Goda, F., Demidenko, E. &Swartz, H. M. Effect on regrowth delay in a murine tumor of schedulingspit dose radiation based on direct pO2 measurements by EPR oximetry.Radia. Res. 150, 549-556, 1998; O'Hara, et al., Response toradioimmunotherapy correlates with tumor pO2 measured by EPR oximetry inhuman tumor xenografts. Radia. Res. 155, 466-473, 2001].

However, conventional targeted radiotherapy methods typically fail toeffective use a high degree of fluorine content and/or a high degree ofsymmetry to provide a mechanism for high sensitivity ¹⁹F NM analysis.

Despite conventional fluorinated compounds and conventional targetedradiotherapy, there remains a need for methods and compositions thatovercome these deficiencies.

SUMMARY

Disclosed are compounds comprising the structure:

wherein p is a non-negative integer; wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂,R₂₃, R₃₁, R₃₂, and R₃₃ are, independently, H, CH₃, CF₃ or alkyl; andwherein R₄ is H, OH, OBn, OC(CF₃)₃, alkyl, or alkoxy.

Also disclosed are compounds comprising the structure:

wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, and R₃₃ are,independently, H, CH₃, CF₃ or alkyl; and wherein R₄ is H, OH, OBn,OC(CF₃)₃, alkyl, or alkoxy.

Also disclosed are compounds comprising a hydrophilic moiety and ahydrophobic moiety, wherein the hydrophobic moiety exhibits maximumsymmetric branching.

Also disclosed are bilayers, micelles, and coatings comprising thedisclosed compounds.

Also disclosed are processes for the preparation of a compoundcomprising the structure:

wherein R is H, CH₃, CF₃ or alkyl and wherein R₄ is H, OH, OBn, alkyl,or alkoxy; the process comprising the steps of: providing a triol,reacting the triol with tert-butanol or nonafluoro-tert-butanol toprovide a tri-tert-butyl ether or a triperfluoro-tert-butyl ether.

Also disclosed are the products produced by the disclosed processes.

Also disclosed are nanoparticles comprising the disclosed compounds.

Also disclosed are multifunctional delivery vehicles comprising at leastone of the disclosed compounds, wherein R₄ comprises a moiety having thestructure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl; and atleast one of the disclosed compounds, wherein R₄ comprises a moietyhaving the structure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; wherein A is O, S, or amino; and whereinR′ comprises a peptide.

Also disclosed are pharmaceutical compositions comprising one or more ofthe disclosed compounds or pharmaceutically acceptable salts or prodrugsthereof, and one or more pharmaceutically acceptable carriers.

Also disclosed are methods for the treatment of a disease ofuncontrolled cellular proliferation comprising administering to a mammaldiagnosed as having a disease of uncontrolled cellular proliferation oneor more of the disclosed compounds, one or more of the disclosednanoparticles, one or more of the disclosed a multifunctional deliveryvehicles, one or more of the disclosed pharmaceutical compositions, orpharmaceutically acceptable salts or prodrugs thereof, in an amounteffective to treat the disease of uncontrolled cellular proliferation.

Also disclosed are compounds comprising the structure:

wherein p is a non-negative integer; wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂,R₂₃, R₃₁, R₃₂, and R₃₃ are, independently, H, CH₃, CF₃ or alkyl; andwherein R₄ comprises the structure:

wherein q is a non-negative integer, and wherein Z comprises CH₂OH,CH₂NH₂, CH₂SH, CO₂H, CH₂O(CH₂CH₂O)₄H, or a substituted or unsubstitutedamide.

Also disclosed are compounds wherein the substituted or unsubstitutedamide comprises the structure:

wherein R is OH, NH₂, NH-alkyl, alkyl, polyalkylene oxide, a moietyhaving the structure:

or a moiety having the structure:

wherein R₇, R₉, and R₉ are, independently, H, CH₂CO₂H, or alkyl, andwherein Ar is an aryl group.

Also disclosed are compounds wherein the substituted or unsubstitutedamide comprises the structure:

wherein b is a non-negative integer, and wherein R is OH, NH₂, NH-alkyl,alkyl, polyalkylene oxide, a moiety having the structure:

or a moiety having the structure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl, andwherein Ar is an aryl group.

Also disclosed are bilayers, micelles, (micro)emulsions, andnanoparticles comprising the disclosed compounds.

Also disclosed are multifunctional delivery vehicles comprising at leastone the disclosed compounds comprising a moiety having the structure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl.

Also disclosed are methods of delivering radionuclides for radiotherapycomprising the steps of complexing a radionuclide with a disclosedcompound comprising a moiety having the structure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl; andadministering the complex to a mammal in an amount effective forradiotherapy.

Also disclosed are methods of delivering a metallic ion for ¹H imagingcomprising the steps of complexing a metal ion with a disclosed compoundcomprising a moiety having the structure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl; andadministering the complex to a subject in an amount effective fordetection by ¹H MRI.

Also disclosed are compounds comprising the structure: R_(φ)−R_(ψ);wherein R_(φ) comprises a branched hydrophobic moiety comprisinghydrocarbon, perfluorocarbon, partial fluorocarbon, or a hybrid analoguewith O, S, N, B, Si, or P, wherein each multivalent atom in R_(φ) has avalency, v, and is unbranched or substituted with v-1 identicalsubstituents, wherein R_(φ) has at least one branching point other thana t-butyl or perfluoro-t-butyl group, wherein the branching point is amultivalent atom having a valency, v, of greater than or equal to 3, andwherein the branching point is substituted with v-1 identicalsubstituents, and wherein the branching point identical substituentseach comprise at least one multivalent atom; and wherein R_(ψ) comprisesan unbranched hydrophilic moiety having the structure:(CH₂)_(i)[Y(CH₂)_(j)]_(k)Z, wherein i, j, and k are, independently,non-negative integers, wherein Y is O, S, or NH, and wherein Z is OH,SH, CO₂H, SO₃H, OP(O)(OH)₂, silyl, a primary amine, a secondary amine, atertiary amine, a quaternary ammonium salt, an amino acid, a peptide, asugar, an (oligo)nucleotide, or a chelator.

Also disclosed are compounds comprising the structure: R_(φ)−R_(ψ);wherein R_(φ) comprises a branched hydrophobic moiety comprisinghydrocarbon, perfluorocarbon, partial fluorocarbon, or a hybrid analoguewith O, S, N, B, Si, or P, wherein each multivalent atom in R_(φ) has avalency, v, and is unbranched or substituted with v-1 identicalsubstituents, wherein R_(φ) has at least one branching point other thana t-butyl or perfluoro-t-butyl group, wherein the branching point is amultivalent atom having a valency, v, of greater than or equal to 3, andwherein the branching point is substituted with v-1 identicalsubstituents, and wherein the branching point identical substituentseach comprise at least one multivalent atom; and wherein R_(ψ) comprisesa branched or unbranched hydrophilic moiety comprising OH, a primaryamine, a secondary amine, a tertiary amine, a quaternary ammonium salt,substituted or unsubstituted alkyl, or substituted or unsubstitutedalkoxy.

Also disclosed are methods of delivering radionuclides for radiotherapycomprising the steps of complexing a radionuclide with at least one ofthe disclosed compound; and administering the complex to a mammal in anamount effective for radiotherapy.

Also disclosed are methods of delivering a metallic ion for ¹H imagingcomprising the steps of complexing a metal ion with at least one of thedisclosed compounds; and administering the complex to a subject in anamount effective for detection by ¹H MRI.

Also disclosed are methods of delivering a fluorocarbon for ¹⁹F imagingcomprising the steps of administering at least one of the disclosedcompounds to a subject in an amount effective for detection by ¹⁹F MRI;and performing a ¹⁹F MRI experiment of the subject.

Also disclosed are methods of delivering oxygen comprising the steps ofcomplexing oxygen with at least one of the disclosed compounds; andadministering the complex to a subject.

Also disclosed are multi-modular treatment methods comprising the stepof simultaneously performing at least two of the disclosed methods.

Additional advantages can be set forth in part in the description whichfollows, and in part can be obvious from the description, or may belearned by practice. Other advantages can be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description serve to explain the principles of the invention.

FIG. 1 shows the ¹⁹F NMR spectrum of an F-oil (compound 5). The signalaround 71 ppm is from the F-oil and the signal around 164 ppm is fromthe standard, hexafluorobenzene.

FIG. 2 shows a schematic of a multifunctional delivery vehicle forimage-guided targeted radiotherapy. The fluorocarbon nanoparticle, thechelators, and the peptide are not drawn in proportion. In the diagram,each nanoparticle carries two chelators and one targeting peptide. Eachnanoparticle can carry multiple chelators and targeting peptides.

FIG. 3 shows microemulsions (and/or nanoemulsions) formed by twodifferent F-surfactants. The F-oil used here was and analog of compound4 with —OBn replaced by -Me; the F-surfactant used in the 1st row wascompound 10, while that used in the 2nd row was an analog of compound 10with eighth (—OCH₂CH₂—) units.

FIG. 4 shows FIG. 4 shows the ¹⁹F NMR spectra of fluorocarbonnanoparticles for one representative spectrum. a. Emulsion formulated byus on a 200 ppm scale. b. The same ¹⁹F spectrum on a 1 ppm scale. TheF-oil and the F-surfactant peaks are very close (0.25 ppm apart); c. ¹⁹Fspectrum of fluorocarbon nanoparticles in one published study ¹⁹F, alsoon a 200 ppm scale. The F-oil used is an analog of compound 4 with —OBnreplaced by -Me, while the F-surfactant is compound 10.

FIG. 5 shows the SAXS analysis of fluorocarbon nanoparticles.

FIG. 6 shows the gradual gelation of a fluorocarbon nanoemulsion as theamount of added F-surfactant increases (the amount of F-surfactantincreases from left to right). The F-oil used in here was an analog ofcompound 4 with —OBn replaced by -Me; the F-surfactant used was compound10.

FIG. 7 shows a schematic representation of the multi-modularfluorocarbon nanoparticles.

FIG. 8 shows (a) singlet ¹⁹F NMR peak of G0-G3 (25° C., 0.025M in CD₃OD,376 MHz) and (b) T1 and T2 increase with molecular weight [T1 & T2 ofG0-G3 (¹⁹F, 25° C., 0.025M in CD₃OD, 376 MHz)].

FIG. 9 shows ¹⁹F NMR spectra of chelator 35 and 1 (0.025 M in CD₃OD, 376MHz).

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which mayneed to be independently confirmed.

A. DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound,” “apolymer,” or “a particle” includes mixtures of two or more suchcompounds, polymers, or particles, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it can beunderstood that the particular value forms another embodiment. It can befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that throughoutthe application, data is provided in a number of different formats andthat this data represents endpoints and starting points, and ranges forany combination of the data points. For example, if a particular datapoint “10” and a particular data point 15 are disclosed, it isunderstood that greater than, greater than or equal to, less than, lessthan or equal to, and equal to 10 and 15 are considered disclosed aswell as between 10 and 15. It is also understood that each unit betweentwo particular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— units in thepolyester, regardless of whether ethylene glycol was used to prepare thepolyester. Similarly, a sebacic acid residue in a polyester refers toone or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Unless explicitly disclosed, this disclosure is notintended to be limited in any manner by the permissible substituents oforganic compounds. Also, the terms “substitution” or “substituted with”include the implicit proviso that such substitution is in accordancewith permitted valence of the substituted atom and the substituent, andthat the substitution results in a stable compound, e.g., a compoundthat does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc.

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

As used herein, the term “alkyl” refers to a hydrocarbon group that canbe conceptually formed from an alkane, alkene, or alkyne by removinghydrogen from the structure of a cyclic or non-cyclic hydrocarboncompound having straight or branched carbon chains, and replacing thehydrogen atom with another atom or organic or inorganic substituentgroup. In some aspects of the invention, the alkyl groups are “C₁ to C₆alkyl” such as methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, amyl, tert-amyl, and hexyl groups, their alkenylanalogues, their alkynyl analogues, and the like. Many embodiments ofthe invention comprise “C₁ to C₄ alkyl” groups (alternatively termed“lower alkyl” groups) that include methyl, ethyl, propyl, iso-propyln-butyl, iso-butyl, sec-butyl, and t-butyl groups, their alkenylanalogues, their alkynyl analogues, or the like. Some of the preferredalkyl groups of the invention have three or more carbon atoms preferably3 to 16 carbon atoms, 4 to 14 carbon atoms, or 6 to 12 carbon atoms. Thealkyl group can be unsubstituted or substituted. A hydrocarbon residue,for example an alkyl group, when described as “substituted,” contains oris substituted with one or more independently selected heteroatoms suchas O, S, N, P, or the halogens (fluorine, chlorine, bromine, andiodine), or one or more substituent groups containing heteroatoms (OH,NH₂, NO₂, SO₃H, and the like) over and above the carbon and hydrogenatoms of the substituent residue. Substituted hydrocarbon residues mayalso contain carbonyl groups, amino groups, hydroxyl groups and thelike, or contain heteroatoms inserted into the “backbone” of thehydrocarbon residue. In one aspect, an “alkyl” group can be fluorinesubstituted. In a further aspect, an “alkyl” group can beperfluorinated.

In certain aspects, the term “alkyl” as used herein is a branched orunbranched saturated hydrocarbon group of 1 to 24 carbon atoms, forexample 1 to 12 carbon atoms or 1 to 6 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl,n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl,decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and thelike. The alkyl group can also be substituted or unsubstituted. Thealkyl group can be substituted with one or more groups including, butnot limited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “loweralkyl” group is an alkyl group containing from one to six carbon atoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, substituted orunsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiolas described herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula —(CH₂)_(a)—, where “a” is an integer of from2 to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹-OA² or—OA¹-(OA²)_(a)-OA³, where “a”, is an integer of from 1 to 200 and A¹,A², and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A¹A⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onecarbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,norbornenyl, and the like. The term “heterocycloalkenyl” is a type ofcycloalkenyl group as defined above, and is included within the meaningof the term “cycloalkenyl,” where at least one of the carbon atoms ofthe ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group andheterocycloalkenyl group can be substituted or unsubstituted. Thecycloalkenyl group and heterocycloalkenyl group can be substituted withone or more groups including, but not limited to, substituted orunsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiolas described herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,substituted or unsubstituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro,silyl, sulfo-oxo, or thiol, as described herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-basedring composed of at least seven carbon atoms and containing at least onecarbon-carbon triple bound. Examples of cycloalkynyl groups include, butare not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and thelike. The term “heterocycloalkynyl” is a type of cycloalkenyl group asdefined above, and is included within the meaning of the term“cycloalkynyl,” where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkynyl group andheterocycloalkynyl group can be substituted or unsubstituted. Thecycloalkynyl group and heterocycloalkynyl group can be substituted withone or more groups including, but not limited to, substituted orunsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiolas described herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, substituted or unsubstituted alkyl, cycloalkyl, alkoxy,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term“biaryl” is a specific type of aryl group and is included in thedefinition of “aryl.” Biaryl refers to two aryl groups that are boundtogether via a fused ring structure, as in naphthalene, or are attachedvia one or more carbon-carbon bonds, as in biphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by theformula NA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen orsubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein. The term “polyester” as usedherein is represented by the formula -(A¹O(O)C-A²-C(O)O)_(a)— or-(A¹O(O)C-A²-OC(O))_(a)—, where A¹ and A² can be, independently, asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and“a” is an integer from 1 to 500. “Polyester” is as the term used todescribe a group that is produced by the reaction between a compoundhaving at least two carboxylic acid groups with a compound having atleast two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, a substituted or unsubstitutedalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group described herein. The term “polyether” as usedherein is represented by the formula -A¹O-A²O)_(a)—, where A¹ and A² canbe, independently, a substituted or unsubstituted alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl groupdescribed herein and “a” is an integer of from 1 to 500. Examples ofpolyether groups include polyethylene oxide, polypropylene oxide, andpolybutylene oxide.

The term “halide” as used herein refers to the halogens fluorine,chlorine, bromine, and iodine. It is also understood that, in certainaspects, pseudohalides (e.g., tosyl or mesyl groups) can be substitutedfor halo groups.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, a substituted or unsubstitutedalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or a substituted orunsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A′, or —OS(O)₂OA¹, where A¹ can be hydrogen ora substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.Throughout this specification “S(O)” is a short hand notation for S═O.The term “sulfonyl” is used herein to refer to the sulfo-oxo grouprepresented by the formula —S(O)₂A′, where A¹ can be hydrogen or asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “sulfone” as used herein is represented by the formulaA¹S(O)₂A², where A¹ and A² can be, independently, a substituted orunsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein. The term“sulfoxide” as used herein is represented by the formula A¹S(O)A², whereA¹ and A² can be, independently, a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture.

Disclosed are the components to be used to prepare the compositions aswell as the compositions themselves to be used within the methodsdisclosed herein. These and other materials are disclosed herein, and itis understood that when combinations, subsets, interactions, groups,etc. of these materials are disclosed that while specific reference ofeach various individual and collective combinations and permutation ofthese compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particular compoundis disclosed and discussed and a number of modifications that can bemade to a number of molecules including the compounds are discussed,specifically contemplated is each and every combination and permutationof the compound and the modifications that are possible unlessspecifically indicated to the contrary. Thus, if a class of molecules A,B, and C are disclosed as well as a class of molecules D, E, and F andan example of a combination molecule, A-D is disclosed, then even ifeach is not individually recited each is individually and collectivelycontemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E,and C-F are considered disclosed. Likewise, any subset or combination ofthese is also disclosed. Thus, for example, the sub-group of A-E, B-F,and C-E would be considered disclosed. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the compositions. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. SYMBOLS AND ABBREVIATIONS

Bn: benzyl; Boc: t-butoxycarbonyl; Bz: benzoyl; CEST: chemical exchangesaturation transfer; Cys: cysteine; DCC: 1,3-dicylclohexylcarbodiimide;DCM: dichloromethane; DEAD: diethylazodicarboxylate; DMAP:4-dimethylaminopyridine; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate; EYP: egg yolkphospholipids; Fmoc: fluorenylmethoxycarbonyl; F-oil: fluorinated oils;F-surfactant: fluorinated surfactants; HPLC: high-performance liquidchromatography; ICP-OES: Inductively coupled plasma optical emissionspectrometry; LC: liquid chromatography; Lys (K): lysine; MRI: magneticresonance imaging; MRS: magnetic resonance spectroscopy; MS: massspectrometry; Ms: methanesulfonyl; NMR: nuclear magnetic resonance; o/w:oil-in-water; PBS: physiological buffer systems; Phe: phenylalanine;pO₂: oxygen partial pressure (also called oxygen tension); SAXS:small-angle X-ray scattering; TBAF: tetra-butylammonium fluoride; TBS:t-butyldimethylsilyl; tBu: t-butyl; TFA: trifluoroacetic acid; THF:tetrahydrofuran; Thr: threonine: Trp: tryptophan; and Tyr: tyrosine.

C. TABLE OF COMPOUNDS

D. HIGHLY FLUORINATED COMPOUNDS

In one aspect, the compounds are highly fluorinated compounds. In afurther aspect, the compounds are partially fluorinated compounds. In aneven further aspect, the compounds are non-fluorinated, yet hydrophobic,compounds.

1. Structure

In one aspect, a compound comprises the structure:

wherein p is a non-negative integer; wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂,R₂₃, R₃₁, R₃₂, and R₃₃ are, independently, H, CH₃, CF₃, or alkyl; andwherein R₄ is H, OH, OBn, OC(CF₃)₃, alkyl, or alkoxy. In a furtheraspect, p is 2, 3, 4, or 5. In one aspect, at least one of R₁₁, R₁₂,R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, or R₃₃ is CF₃. In a further aspect, R₁₁,R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, and R₃₃ are CF₃.

In a yet further aspect, a compound comprises the structure:

wherein p is a non-negative integer; wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂,R₂₃, R₃₁, R₃₂, and R₃₃ are, independently, H, CH₃, CF₃, or alkyl; andwherein R₄ is H, OH, OBn, OC(CF₃)₃, alkyl, or alkoxy. In one aspect, atleast one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, or R₃₃ is CF₃. In afurther aspect, R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, and R₃₃ are CF₃.

In a further aspect, a compound comprises the structure:

wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, and R₃₃ are,independently, H, CH₃, CF₃, or alkyl; and wherein R₄ is H, OH, OBn,OC(CF₃)₃, alkyl, or alkoxy. In a further aspect, R₁₁, R₁₂, and R₁₃ areCF₃. In a further aspect, R₂₁, R₂₂, and R₂₃ are CF₃. In a furtheraspect, R₃₁, R₃₂, and R₃₃ are CF₃. In one aspect, at least one of R₁₁,R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, or R₃₃ is CF₃. In a further aspect,R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, and R₃₃ are CF₃.

In one aspect, the compound is a surfactant. In a further aspect, thecompound comprises a structure:

In a further aspect, the compound exhibits maximum symmetric branching.

In one aspect, the compound is an oil. In a further aspect, the compoundcomprises a structure:

In a further aspect, the compound exhibits maximum symmetric branching.

In one aspect, R₄ comprises the structure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; wherein A is O, S, or amino; and whereinR′ comprises H, CH₂CO₂H, silyl, alkyl, or a peptide.

In a further aspect, R₄ comprises the structure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; and wherein R₇, R₈, and R₉ are,independently, H, CH₂CO₂H, or alkyl.

In one aspect, a compound comprises a hydrophilic moiety and ahydrophobic moiety, wherein the hydrophobic moiety exhibits maximumsymmetric branching.

In one aspect, a compound comprises the structure:R_(φ)−R_(ψ);wherein R_(φ) comprises a branched hydrophobic moiety comprisinghydrocarbon, perfluorocarbon, partial fluorocarbon, or a hybrid analoguewith O, S, N, B, Si, or P, wherein each multivalent atom in R_(φ) has avalency, v, and is branched or unbranched, wherein R_(φ) has at leastone branching point other than a t-butyl or perfluoro-t-butyl group,wherein the branching point is a multivalent atom having a valency, v,of greater than or equal to 3, and wherein the branching point issubstituted with v-1 or v-2 identical substituents if v=4 or substitutedwith v-1 identical substituents if v=3, and wherein the branching pointidentical substituents each comprise at least one multivalent atom; andwherein R_(ψ) comprises an unbranched hydrophilic moiety having thestructure:(CH₂)_(i)[Y(CH₂)_(j)]_(k)Z,wherein i, j, and k are, independently, non-negative integers, wherein Yis O, S, or NH, and wherein Z is OH, SH, CO₂H, SO₃H, OP(O)(OH)₂, silyl,a primary amine, a secondary amine, a tertiary amine, a quaternaryammonium salt, an amino acid, a peptide, a sugar, an (oligo)nucleotide,or a chelator. The compound can be a surfactant.

In a further aspect, a compound comprises the structure:R_(φ)−R_(ψ);wherein R_(φ) comprises a branched hydrophobic moiety comprisinghydrocarbon, perfluorocarbon, partial fluorocarbon, or a hybrid analoguewith O, S, N, B, Si, or P, wherein each multivalent atom in R_(φ) has avalency, v, and is branched or unbranched, wherein R_(φ) has at leastone branching point other than a t-butyl or perfluoro-t-butyl group,wherein the branching point is a multivalent atom having a valency, v,of greater than or equal to 3, and wherein the branching point issubstituted with v-1 identical substituents, and wherein the branchingpoint identical substituents each comprise at least one multivalentatom; and wherein R_(φ) comprises a branched or unbranched hydrophilicmoiety comprising OH, a primary amine, a secondary amine, a tertiaryamine, a quaternary ammonium salt, substituted or unsubstituted alkyl,or substituted or unsubstituted alkoxy. The compound can be an oil.

In one aspect, a compound comprises the structure:

wherein p is a non-negative integer; wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂,R₂₃, R₃₁, R₃₂, and R₃₃ are, independently, H, CH₃, CF₃ or alkyl; andwherein R₄ comprises the structure:

wherein q is a non-negative integer, and wherein Z comprises CH₂OH,CH₂NH₂, CH₂SH, CO₂H, CH₂—O—(CH₂CH₂O)_(j)H, or a substituted orunsubstituted amide, wherein j is a positive integer. In a furtheraspect, p and q are, independently, 2, 3, 4, or 5. In a further aspect,R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, and R₃₃ are CF₃. In a furtheraspect, p is 0 and the compound comprises the structure:

In a further aspect, R₄ comprises the structure:

wherein q is a non-negative integer, and wherein Z comprises CH₂OH,CH₂NH₂, CH₂SH, CO₂H, or CH₂—O—(CH₂CH₂O)₄H. In a yet further aspect, Zcomprises CH₂OH, CH₂NH₂, CH₂SH, CO₂H, or CH₂—O—(CH₂CH₂O)₄H. In a furtheraspect, q is 3.

In a further aspect, R₄ comprises the structure:

wherein q is a non-negative integer, and wherein Z comprises asubstituted or unsubstituted amide.

In one aspect, the substituted or unsubstituted amide comprises thestructure:

wherein R is OH, NH₂, NH-alkyl, alkyl, polyalkylene oxide, a moietyhaving the structure:

or a moiety having the structure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl, andwherein Ar is an aryl group.

In a further aspect, R comprises the structure:

In a further aspect, R comprises the structure:

In one aspect, the substituted or unsubstituted amide comprises thestructure:

wherein b is a non-negative integer, and wherein R is OH, NH₂, NH-alkyl,alkyl, polyalkylene oxide, a moiety having the structure:

or a moiety having the structure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl, andwherein Ar is an aryl group. In a further aspect, b is 0, 1, 2, or 3.

In one aspect, R comprises the structure:

In a further aspect, R comprises the structure:

a. Chelator

The macrocyclic chelator DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), for example, canbe used as the metallic ion carrier in the present compounds becauseion-DOTA complexes have thermodynamic and kinetic stability [Bianchi, etal., Thermodynamic and structural properties of Gd(III) complexes withpolyamino-polycarboxylic ligands: basic compounds for the development ofMRI contrast agents. Coord. Chem. Rev. 204, 309-393, 2000]. DOTA is thechelator used in the radio-therapeutic drug OctreoTher® for ⁹⁰Y³⁺complexation and the MRI contrast agent Dotarem® for Gd³⁺ complexation.Analogs of DOTA or other chelators (e.g., DTPA) can also be used.

b. Targeting Peptide

Octreotide can be used, for example, as the targeting for the presentcompounds. Octreotide is an octapeptide analog of the natural hormonesomatostatin and has been approved for treating acromegaly(Sandostatin®). It is also the targeting peptide used in theradiodiagnostic agent OctreoScan® (FDA-approved) and radiotherapeuticagent OctreoTher® (under clinical trials). Hence it has a proven recordfor clinical use. It targets neuroendocrine tumors that over-expresstype 2 stamotostatin receptors (sstr₂) [Kaltsas, G. A., Papadogias, D.,Makras, P. & Grossman, A. B. Treatment of advanced neuroendocrinetumours with radiolabelled somatostatin analogues. Endocrine-RelatedCancer, 12, 683-699, 2005]. Other analogs of octreotide (such as, butnot restricted to, lanreotide, vapreotide, etc.) can be used for thispurpose as well. Other peptides (such as, but not restricted to,bombesin, vascoactive intestinal peptide, cholecystokinin, substance P,etc.) can also be used for this purpose (L. Bodei, G. Paganelli & G.Mariani, Receptor radionuclide therapy of tumors: a road from basicresearch to clinical applications. J. Nucl. Med. 47, 375-377, 2006).

2. Maximum Symmetric Branching

In one aspect, each compound can have a hydrophilic moiety and ahydrophobic moiety. The hydrophilic moiety can be an unbranched chain ofvariable length and with different terminal groups. The chemical natureof the hydrophilic chain can be variable (e.g., oxyethylene units,oxypropylene units, etc.). In one aspect, the hydrophobic moiety isbranched and satisfies the principle of “maximum symmetric branching.”In a one aspect, at any point in the hydrophobic moiety, there is eitherno branching (i.e., —CH₂—, —CF₂—, —NH—, —BH—, —O—, —S—) or has maximumnumber of branches with all branches identical (e.g., a carbon or asilicon should have three identical branches and a nitrogen or a boronshould have two identical branches). The hydrophobic moiety can containat least one branching point. A terminal perfluoro-tert-butyl group istypically not counted as a branching point.

In one aspect, every branch point can be symmetric and at least onebranching point is maximally symmetric, with the stipulation thatterminal tert-butyl or perfluoro-tert-butyl cannot be the sole maximallysymmetric branching point in a molecule.

In a sense, this describes a chain growth mechanism for the surfactantor oil molecule. The resultant surfactant or oil molecules can be“umbrella-shaped” with the hydrophilic and the hydrophobic moietiesbeing the “pole” and the canopy of the “umbrella,” respectively. As aresult of symmetric branching, terminal groups in the hydrophobic moietyhave, in one aspect, perfect spherical symmetry, leading to a single ¹⁹Fsignal for magnetic resonance detection. As a result of maximumbranching, the surfactant has, in one aspect, maximum terminal surfacearea for a given number of main-chain atoms. This gives the best surfaceprotection if these surfactants are used as, for example, coatingmaterials.

3. Surfactants

In one aspect, the compounds are surfactants having a hydrophobic moietyand a hydrophilic moiety. Exemplary surfactants include the followingstructures.

4. Oils

In one aspect, the compounds are oils having at least one hydrophobicmoiety. Exemplary oils include the following structures.

E. METHODS OF PREPARATION

In one aspect, the methods relate to a process for the preparation of acompound comprising the structure:

wherein R is H, CH₃, CF₃, or alkyl and wherein R₄ is H, OH, OBn, alkyl,or alkoxy; the process comprising the steps of: providing a triol,reacting the triol with tert-butanol or nonafluoro-tert-butanol toprovide a tri-tert-butyl ether or a triperfluoro-tert-butyl ether. In afurther aspect, the reacting step is performed withnonafluoro-tert-butanol. In a further aspect, the triol ispentraerythritol, mono-silylated pentraerythritol, or2,2-bis-hydroxymethyl-propan-1-ol. In one aspect, R₄ comprises methyl,ethyl, n-propyl, isopropyl, or butyl. In one aspect, R is CF₃.

In one aspect, the providing step is performed by the steps of:mono-protecting pentraerythritol before the reacting step, anddeprotecting the product of the reacting step. In a further aspect, thereacting step occurs before the deprotecting step. In a further aspect,the process can further comprise the step of coupling the product thedeprotecting step with a hydrophilic compound.

In one aspect, the hydrophilic compound comprises a moiety having thestructure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; wherein R′ comprises H, CH₂CO₂H, silyl,or alkyl; wherein A is O, S, or amino; and wherein X is a leaving group.In one aspect, n is an integer from 4 to 12. In a further aspect, theprocess further comprises the step of cleaving the silyl group. In afurther aspect, the process further comprises the step of conjugatingwith cyclen or a compound comprising a cyclen residue.

In a further aspect, the compound comprises the structure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; and wherein R₇, R₈, and R₉ are,independently, H, CH₂CO₂H, or alkyl. In a further aspect, R is CF₃. In afurther aspect, n is an integer from 4 to 12.

In one aspect, the compounds and compositions relate to the product(s)produced by the process(es) of the methods.

One example highly fluorinated compound, 1 F-DOTA, was prepared by themethods disclosed herein:

The synthesis of 1 F-DOTA started with the modification ofpentaerythritol. Protection pentaerythritol 2 as the orthoacetate,followed by protection of the fourth hydroxyl group with benzyl bromideand hydrolysis of the orthoacetate, furnished the corresponding triol 3[Dunn, T. J.; Neumann, W. L.; Rogic, M. M.; Woulfe, S. R. J. Org. Chem.1990, 55, 6368-6373.]. Then, triol 3 was reacted withnonafluoro-tert-butanol in the presence of diethylazodicarboxylate,triphenylphosphine and powdered 4 Å molecular sieve to provide theperfluoro-tert-butyl ether 4 in one step with good yield.Tri-perfluoro-tert-butyl ether 4 was isolated from the reaction mixtureby simple F362 extraction, and no mono- or di-perfluoro-tert-butyl etherwas detected from the extract. Lewis acid (aluminum chloride) mediatedremoval of the benzyl ether completed construction of the desired highlyfluorinated alcohol 5 in substantially quantitative yield on 50 gramscale.

To prepare the linker 8, one of the hydroxyl groups in the tetraethyleneglycol 6 was selectively protected as tert-butyldimethylsilyl ether 7,which was then treated with methanesulfonyl chloride and triethylamineto give the linker 8.

Alternately, linker 8′ can be prepared. One of the hydroxyl groups inthe tetraethylene glycol 6 can be selectively protected as benzyl ether7′, which can then be treated with methanesulfonyl chloride andtriethylamine to give the linker 8′.

The conjugation of DOTA with fluorinated alcohol 5 was then performed.The highly fluorinated alcohol 5 was first attached to the hydrophilicchain to provide compound 9 in good yield by treating the alcohol 5 withpotassium hydride in tetrahydrofuran at room temperature for 30 minutesand then slowly adding methanesulfonylate 8 (or, alternatively, 8′) atthe same temperature. Due to the three bulky perfluoro-tert-butyl groupin compound 5, sodium hydride failed to deprotonate the hydroxyl groupin alcohol 5 and resulted in recovery of the alcohol 5 andmethanesulfonylate 8 after a long reaction time. After removal of thetert-butyldimethylsilyl group in compound 9 by tetrabutylammoniumfluoride, the newly formed hydroxyl group in compound 10 was treatedwith ethanesulfonyl chloride and triethylamine to give themethanesulfonylate 11 in good yield. Because of their surfactantproperties, compounds 9, 10 and 11 typically cannot be extracted fromthe reaction mixtures by F362 (perfluorohexanes), but those compoundscan be easily purified by flash chromatography. Attaching the cyclenring to the fluorinated methanesulfonylate 11 was achieved by treatingcompound 11 with 2 equivalents of cyclen at 60° C. Fluorinated silicagel was used to purify the cyclen derivative 12. The other three “arms”were incorporated into compound 12 by treating 12 with ethylbromoacetatein the presence of potassium carbonate. The resulting ester 13 was thenhydrolyzed to give the final product 1.

The synthesis of F-surfactant-octreotide, or any other peptide, anF-surfactant with a carboxylic end group (—COOH) can be used. Such asurfactant can be conjugated to the N-terminus of a peptide as the laststep of solid-phase synthesis, as illustrated below:

Synthesis route of F-surfactant-octreotide (applicable to other peptidesor any molecule which contains an unprotected amine group on asolid-phase support). a. Jones reagent, Acetone, rt.; b. HOBt, DIC, DMF,rt. Octreotide sequence:DPhe1-c[Cys2-Phe3-DTrp-4-Lys5-Thr6-Cys7]-Thr8-NH₂. Standard solid-phasesynthesis using Fmoc chemistry can be employed. The cyclic S—S bondbetween Cys2 and Cys7 can be formed via air oxidation duringpost-cleavage workups.

F. METHODS OF USING

1. Conjugating F-Surfactants to Proteins and Monoclonal Antibodies

If the protein or monoclonal antibodies (mAb) contains thiol group(s),then the F-surfactant with a thiol (—SH) end group can be used to form adisulfide bond, using established S—S bond formation conditions (e.g.,I. Annis, B. Hargittai & G. Barany. Disulfide bond formation inPeptides. Methods in Enzymology, 289, 198-221, 1997; Tam, J. P., Wu,C.-R., Liu, W. & Zhang, J-W. Disulfide bond formation in peptides bydimethyl sulfoxide. Scope and applications. J. Am. Chem. Soc. 113,6657-6662, 1991):F-surfactant-SH+HS-Protein=F-surfactant-S—S-Protein

If the protein or mAb contains thiol group(s), then one alternativemethod is to conjugate it to the F-surfactant with an amine (—NH₂) endgroup, using the commercially available heterobifunctional crosslinkingagent succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate(SMCC) (Bieniarz, C., Husain, M., Barnes, G., King, C. A. & Welch, C. J.Extended length heterofunctional coupling agents for proteinconjugations. Bioconjugate Chem. 8, 88-95, 1996; Weiden, P. L. et al.,Pretargeted radioimmunotherapy for treatment of non-Hodgkin's lymphoma(NHL): initial phase I/II study result. Cancer Biother. & Radiopharm.15, 15-29, 2000; Subbiah, K. et al., Comparison of immunoscintigraphy,efficacy, and toxicity of conventional and pretargetedradioimmunotherapy in CD20-expressing human lymphoma xenografts. J.Nucl. Med. 44, 437-445, 2003). Specifically, one side of SMCC will beconjugated to F-surfactant-NH₂ and the other side will be conjugated tothe thiol group(s) on the protein or antibody, as illustrated in thefollowing scheme:

If the protein or mAb contains amine group(s), then it can be conjugatedto the F-surfactant with a thiol (—SH) end group, using the commerciallyavailable heterobifunctional crosslinking agent succinimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) (Bieniarz, C.,Husain, M., Barnes, G., King, C. A. & Welch, C. J. Extended lengthheterofunctional coupling agents for protein conjugations. BioconjugateChem. 8, 88-95, 1996; Weiden, P. L. et al., Pretargetedradioimmunotherapy for treatment of non-Hodgkin's lymphoma (NHL):initial phase I/II study result. Cancer Biother. & Radiopharm. 15,15-29, 2000; Subbiah, K. et al., Comparison of immunoscintigraphy,efficacy, and toxicity of conventional and pretargetedradioimmuno-therapy in CD20-expressing human lymphoma xenografts. J.Nucl. Med. 44, 437-445, 2003). Specifically, one side of SMCC can beconjugated to F-surfactant-SH and the other side can be conjugated toamine group(s) on the protein or antibody, as illustrated in thefollowing scheme:

2. Methods of Treatment

In one aspect, the methods relate to the treatment of a disease ofuncontrolled cellular proliferation comprising administering to a mammaldiagnosed as having a disease of uncontrolled cellular proliferation oneor more of the disclosed compounds, one or more of the disclosednanoparticles, one or more of the disclosed multifunctional deliveryvehicles, one or more of the disclosed pharmaceutical compositions, orpharmaceutically acceptable salts or prodrugs thereof, in an amounteffective to treat the disease of uncontrolled cellular proliferation.In a further aspect, the disease of uncontrolled proliferation is acarcinoma, lymphoma, leukemia, or sarcoma or other cancers and tumors.In a further aspect, the mammal is a human.

3. Textile-Finishing Agent

Fluorinated textile-finishing agents have been commercialized. Textilestreated with such agent shows excellent both water and oil repellency.There are also some reports indicating that fluorinated surfactants showdurable antimicrobial activity. [Shao, H.; Meng, W.-D.; Qing, F.-L.Synthesis and surface antimicrobial activity of a novelperfluorooctylated quaternary ammonium silane coupling agent. J.Fluorine Chem. 125, 2004, 721-724]. However, reported fluorinatedtextile-finishing agents employed a non-branched perfluorocarbon chain,which means more such agent should be used on textiles to achieveadequate surface properties. Fluorinated surfactants synthesized hereincan have an umbrella shape, making these surfactants are more effectivethan conventional ones.

The surfactant can be cross-linked to the fiber of textile with theending group (such as carboxylic group, or silane group) by followingstandard procedures. [AATCC Technical Manual, American Association ofTextile Chemists and colorists, Research Triangle Park, N.C., 1989.] Theantimicrobial activity and water and oil repellency property can also bedetermined by the standard procedures.

4. Surface Modification Coating

The fluorinated surfactants used as surface coatings can have advantagesover conventional coatings: 1. The fluorinated surfactant can exhibitspecial surface properties, such as, water and oil repellency,antimicrobial, and/or anti-erosion; 2. The fluorinated surfactant canhave an umbrella shape, so the surfactant can exhibit far higherefficiency than common coatings; 3. The surfactant can be conjugated tothe surface by chemical bonds through reaction between the surfactantand the material to be coated, so the coating can be more stable evenunder very extreme conditions than non-bond coatings; 4. By manipulationthe length of the hydrophilic chain, one can easily coat the surface ina three dimensional fashion. [Ulman, A. Formation and Structure ofSelf-Assembled Monolayers Chem. Rev. 1996, 96, 1533-1554]

5. Gene Delivery

One feature in the disclosed designs is a fluorocarbon moiety with allfluorine atoms in spherically symmetric positions, as a result ofsymmetric branching. The consequence of this is that all the fluorineatoms give one ¹⁹F signal (FIG. 1). This can greatly elevate thesensitivity for magnetic resonance detection of the ¹⁹F signal. Such amoiety can be attached to an arbitrary chemical construct (proteins,nucleic acids, polymers, surfactants, oils, etc.) as a ¹⁹F probe. Themuch broader chemical shift range of ¹⁹F (>200 ppm) in comparison withthat of ¹H (ca. 20 ppm) makes it very sensitive toward local chemicalenvironment changes. This has broad implications in analytical,bioanalytical, clinical, and pharmaceutical chemistry. Further, for invivo applications, because there is no MRI-detectable endogenous ¹⁹Fsignal, such a fluorocarbon moiety serves as an excellent probe for invivo biochemical processes associated with normal physiological andpathophysiological states. One specific example is to attach such amoiety to existing nucleic acid condensing agents for gene delivery. Theutility of such a probe is that the chemical shift change of ¹⁹F cansignal the release of encapsulated DNA or RNA, allowing researchers andin the future, clinicians, to determine exactly when the gene isreleased, hence gathering valuable pharmacokinetic data in anon-invasive manner in real time. Such a real-time gathering ofpharmacokinetic data is currently not available. The following diagramillustrating the attachment of an exemplary fluorocarbon moiety to aGemini surfactant currently explored as gene delivery agent [Kirby, A.;Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. S. M.;Soderman, N, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; Rodriguez, C.L. G.; Guedat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; vanEijk, M. C. P. Angew. Chem. Int. Ed. 2003, 42, 1448-1457].

Current Gemini surfactant structure (no ¹⁹F labeling)

¹⁹F-labeled Gemini surfactant structure

6. Multi-Modular Methods

The present multifunctional delivery vehicles can have multiple deliverycapacities, including the delivery of radionuclides for radiotherapy(e.g., ⁹⁰Y³⁺) and/or nuclear imaging (¹¹¹In³⁺ or ⁸⁶Y³⁺), the deliverystable metallic ions for ¹H MR imaging (e.g., Gd³⁺ or Tb³⁺), thedelivery of fluorocarbons for ¹⁹F MR imaging, ¹⁹F oximetry and eventemperature measurement, the delivery of O₂, the delivery ofchemo-drugs, the delivery of chemo- and radio-therapy adjuvants, thedelivery of nucleic acids (DNA, RNA) for gene therapy, etc. However, inone aspect, the delivery vehicle does not have to delivery everythingsimultaneously. It can delivery one, two, three, four five, or more ofthese agents. Due to the modular design of the nanoparticles, multiplefunctions can be embedded into one delivery vehicle through theincorporation of multiple modules. For examples of the multi-modularmethods using the present compounds, see FIG. 7.

In one aspect, the methods relate to delivering radionuclides forradiotherapy comprising the steps of complexing a radionuclide with atleast one of the present compounds; and administering the complex to amammal in an amount effective for radiotherapy. In a further aspect, theradionuclide is at least one of ¹¹¹In³⁺ or ⁸⁶Y³⁺.

In one aspect, the methods relate to A method of delivering a metallicion for ¹H imaging comprising the steps of complexing a metal ion withat least one of the present compounds; and administering the complex toa subject in an amount effective for detection by ¹H MRI. In a furtheraspect, the ion is at least one of Gd³⁺ or Tb³⁺.

In one aspect, the methods relate to delivering a fluorocarbon for ¹⁹Fimaging comprising the steps of administering at least one of thepresent compounds to a subject in an amount effective for detection by¹⁹F MRI; and performing a ¹⁹F MRI experiment of the subject. In afurther aspect, the subject is a mammal, for example, a human.

In one aspect, the methods relate to delivering oxygen comprising thesteps of complexing oxygen with at least one of the present compounds;and administering the complex to a subject.

In one aspect, the methods relate to a multi-modular treatment methodcomprising the step of simultaneously performing at least two of thepresent methods. In a further aspect, at least three of the presentmethods are simultaneously performed.

G. COMPOSITIONS

In one aspect, the compositions relate to a bilayer comprising at leasttwo molecules of the disclosed compounds. In a further aspect, thecompositions relate to a micelle comprising a plurality of molecules ofthe disclosed compounds. In a further aspect, the compositions relate toa coating comprising at least one molecule of the disclosed compounds.In a further aspect, the compositions relate to a pharmaceuticalcomposition comprising one or more of the disclosed compounds orpharmaceutically acceptable salts or prodrugs thereof, and one or morepharmaceutically acceptable carriers.

H. KITS

Disclosed herein are kits that are drawn to compounds and/or reagentsthat can be used in practicing the methods disclosed herein. The kitscan include any reagent or combination of reagents discussed herein orthat would be understood to be required or beneficial in the practice ofthe disclosed methods. For example, the kits could include reagents toperform complexation reactions discussed in certain embodiments of themethods, as well as buffers and solvents required to use the reagents asintended.

I. COMPOSITIONS WITH SIMILAR FUNCTIONS

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures which can perform the same function which arerelated to the disclosed structures, and that these structures willultimately achieve the same result.

J. ¹H-¹⁹F DUAL NUCLEI IMAGING

The combination of high sensitivity and lack of background interferencemakes ¹⁹F an idea candidate for providing an additional MR modalitycomplementary to ¹H MRI. Here, a novel multi-modality MRI regent whichhas a DOTA part for ¹H MRI and a highly branched fluorinated part for¹⁹F MRI was designed and successfully synthesized. All the fluorineatoms in the molecule show only one sharp ¹⁹F NMR signal, therebyindicating that such molecules are ideal for ¹⁹F MRI.

The combination of high sensitivity and lack of background interferencemakes ¹⁹F an idea candidate for providing an additional MR modalitycomplementary to ¹H MRI. Conventional MRI is achieved through the ¹H₂Osignal. Although using ¹H₂O signal for detection affords high resolutionMRI, because of high water concentration in the tissue providessufficient signals, it lacks of specificity. In contrast to the abundantpresence of ¹H₂O, there is no MRI-detectable endogenous ¹⁹F compound inhuman tissues. Therefore, background or interfering endogenous ¹⁹Fsignal is typically negligible. The nuclear magnetic resonancesensitivity of ¹⁹F is only secondary to ¹H (83% as sensitive).

Quantification of in vivo concentration based on ¹H₂O relaxationparameters is typically not reliable due to the heterogeneity of in vivoproton signals [Morawski, A. M.; Winter, P. M.; Crowder, K. C.;Caruthers, S. D.; Fuhrhop, R. W.; Scott, M. J.; Robertson, J. D.;Abendschein, D. R.; Lanza, G. M.; Wickline, S. A. Magn. Reson. Med.2004, 51, 480-486. Morawski, A. M.; Winter, P. M.; Yu, X.; Fuhrhop, R.W.; Scott, M. J.; Hockett, F.; Robertson, J. D.; Gaffney, P. J.; Lanza,G. M.; Wickline, S. A. Magn. Reson. Med. 2004, 52, 1255-1262.]. The lackof background interference makes ¹⁹F MRI an ideal imaging modality forthis application if the drug contains fluorine. [Lanza, G. M.; Yu, X.;Winter, P. M.; Abendschein, D. R.; Karukstis, K. K.; Scott, M. J.;Chinen, L. K.; Fuhrhop, R. W.; Scherrer Morawski, A. M.; Hockett, F.;Robertson, J. D.; Gaffney, P. J.; Wickline, S. A. Magn. Reson. Med.2004, 52, 1255-1262.]. A ¹H-¹⁹F dual nuclei imaging agent allows one toquickly identify areas of interest based on Gd(M)-enhanced ¹H₂O signaland then make more accurate quantification based on the ¹⁹F signal. Whenconjugated to a drug, such a dual nuclei imaging agent allows one todetermine local drug concentration. This enables one to determine theactual amount of drug delivered to pathological sites and hence helps aphysician to develop an image-based dosing for each patient(individualized dosimetry) [Wickline, S. A.; Lanza, G. M. J. Cell.Biochem. (suppl.) 2002, 39, 90-97.].

Multinuclear magnetic resonance imaging (MRI) is playing an increasinglyimportant role in cancer research and holds great potential for cancerdiagnosis and intervention [Gimi, B.; Pathak, A. P.; Ackerstaff, E.;Glunde, K.; Artemov, D.; Bhujwalla, Z. M. Proc. IEEE, 2005, 93,784-799.]. Compared with dual modality imaging methods, such asMRI-optical. MRI-PET or MRI-ultrasound, ¹H-¹⁹F dual nuclei imagingrequires only one imaging modality and is fully compatible with existingMR scanners (magnet, console, etc.) available in almost every hospitalin developed countries; the ¹⁹F signal obtained from ¹⁹F MR spectroscopyor imaging can be overlaid with ¹H MR images (acquired in the sameimaging session) without additional co-registration. Therefore, ¹H-¹⁹Fdual nuclei imaging imposes hardly any extra burden on either thepatients or the health care system.

However, ¹⁹F MRI is usually hampered by the fact that mostperfluorocarbons exhibit multipeak-spectra resulting in ¹⁹F MRI withchemical shift artifacts. On the other hand, for quantitative ¹⁹F MRI,only one resonance peak may be used. In most ¹⁹F MRI research, only veryfew fluorine atoms (10-20% of the total fluorine atoms) have been usedduring the ¹⁹F MRI process. The disclosed highly fluorinated compoundsare suitable for methods employing ¹⁹F MRI because (1) the cyclicchelators, DOTA and its analogs (e.g, DO3A), have the highest stabilitytoward Gd(M) among the chelators currently in clinical use, (2)perfluoro-tert-butyl ether has a very high ¹⁹F signal intensity (27chemical identical fluorine atoms; see FIG. 1) and high stability(stable to base and acid), and (3) tetraethylene glycol has goodbiocompatibility and aqueous solubility.

Fluorinated DOTA and its analogs (collectively referred as F-DOTA) canbe used as imaging agents for the liver and spleen due to preferentialaccumulation of fluorocarbons in these organs.

K. TARGETED FLUOROCARBON NANOPARTICLES

The multifunctional delivery vehicles can be based on fluorocarbonnanoparticles, formulated as microemulsions (also called nanoemulsions).Chelators, peptides and other targeting molecules (e.g, antibodies) canbe attached to the surface of the nanoparticles for radionuclidecarrying and tumor targeting, respectively. The nanoparticles can beassembled in a modular fashion from highly fluorinated molecules inthree steps.

1. Fluorocarbon Nanoparticles

This step can involve the syntheses of highly fluorinated oils (F-oil)and surfactants (F-surfactant) and the formulation of F-oils andF-surfactants into microemulsions of fluorocarbon nanoparticles. Thenanoparticles can have two modules: F-oils and F-surfactants.

2. Chelator-Decorated Fluorocarbon Nanoparticles

A macrocyclic chelator,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA), and/or itsanalogs, can be linked to the end of an F-surfactant, forming a newsurfactant: F-surfactant-DOTA. F-surfactant-DOTA can be added as anotheringredient in microemulsion formulation, forming chelator-coatedfluorocarbon nanoparticles. These nanoparticles can have three modules:F-oil, F-surfactant and F-surfactant-DOTA.

Typically, DOTA forms very stable complexes with trivalent metallic ionsand is the chelator used in the radio-pharmaceutical OctreoTher® (for⁹⁰Y³⁺ chelation) and in the MRI contrast agent Dotarem® (for Gd³⁺chelation). DOTA can be used to chelate Gd³⁺ for ¹H MR imaging. Onepurpose of ¹H-¹⁹F dual imaging is to use these two imaging modes tocorroborate each other. In actual targeted radiotherapy, DOTA can beused to chelate metallic radionuclide ions, such as ⁹⁰Y(III), for cancertreatment.

3. Development of Peptide- and Chelator-Decorated FluorocarbonNanoparticles

A tumor targeting peptide, octreotide, can be conjugated to anF-surfactant, forming a new surfactant: F-surfactant-peptide.F-surfactant-peptide can be added as yet another ingredient inmicroemulsion formulation, forming peptide- and chelator-decoratedfluorous nanoparticles. In one aspect, these nanoparticles can have fourmodules: F-oil, F-surfactant, F-surfactant-DOTA andF-surfactant-peptide.

Octreotide is an octapeptide analog of the natural tetradecapeptidehormone somatostatin and targets neuroendocrine tumors (e.g., pancreaticcancer, small cell lung cancer, etc.). Octreotide is the targetingmoiety in two radiopharmaceuticals: OctreoScan® (FDA-approved), whichcarries ¹¹¹In³⁺ for cancer radio-diagnosis, and OctreoTher® (underclinical trial), which carries ⁹⁰Y³⁺ for cancer radiotherapy.

4. Biodistribution

The biodistribution of targeted fluorocarbon nanoparticles can beconducted in rats with subcutaneous pancreatic tumor implants. Theamount of nanoparticles accumulated in the tumor and in liver and spleencan be determined to calculate the tumor-to-organ ratio. Thetumor-to-organ ratios of targeted versus non-targeted nanoparticles canbe compared to evaluate the effectiveness of targeting.

5. Multifunctional Delivery Vehicles for Image-Guided TargetedRadiotherapy

A multifunctional delivery vehicle can improve the efficacy of targetedradiotherapy in two ways. First, it can tailor the dosing schedule tomeet the pharmacokinetic and pO₂ profiles of each patient (i.e.,individualized treatment plans). Second, it can improve thepharmacokinetic and pO₂ profiles of each patient. The prerequisite forachieving these two goals is to obtain patient-specific pharmacokineticand pO₂ information. Magnetic resonance imaging (MR) is an excellenttool for obtaining patient-specific information for drug deliveryapplications [Swartz, H. Seeing is believing-visualizing drug deliveryin vitro and in vivo. Adv. Drug Del. Rev. 57, 1085-1086, 2005; Kelloff,et al., The progress and promise of molecular imaging probes inoncologic drug development. Clin. Cancer Res. 11, 7967-7985, 2005]. Thedisclosed compounds, methods, and compositions integrate imagingtechnologies and delivery technologies into one multifunctional deliveryvehicle. Such a delivery vehicle can obtain patient-specificpharmacokinetics and pO₂ profiles via MRI and improve thepharmacokinetic and pO₂ profiles of a patient for radiotherapy (e.g., itcan deliver O₂ to the tumor). MRI can then guide the delivery ofradiotherapeutic drugs (image-guided targeted radiotherapy).

The present compositions integrate five roles, radionuclide carrier,tumor targeting, imaging agent, pO₂ probe and O₂ carrier, into onemultifunctional delivery vehicle. The radionuclide carrier function canensure that there are no freely floating radionuclides as they can causesevere radiation damage to normal tissues. The tumor targeting functionensures that the radionuclides will accumulate preferentially in thetumor. The imaging agent function can allow a physician to trace thedrug and visualize the tumor via MRI. Through drug tracing and tumorvisualization that that one can obtain patient-specific pharmacokineticdata and confirm drug targeting. The pO₂ probe function can allow aphysician to determine the oxygen tension at a particular tumor site.pO₂ can serve as a prognostic factor for radiotherapy [Nordsmark, M. &Overgaard, J. A confirmatory prognostic study on oxygenation status andloco-regional control in advanced head and neck squamous cell carcinomatreated by radiation therapy. Radiother. Oncol. 57, 39-43, 2000; Fyles,et al., Oxygenation predicts radiation response and survival in patientswith cervix cancer. Radiother. Oncology, 48, 149-156, 1998]. O₂ deliveryto tumor sites can sensitize hypoxic tumor cells toward radiation withO₂ acts essentially as an adjuvant for radiotherapy [Rockwell, S., Useof a perfluorochemical emulsion to improve oxygenation in a solid tumor.Int. J. Radiation Oncology Biol. Phys. 11, 97-103, 1985; Teicher, B. A.& Rose, C. M. Oxygen-carrying perfluorochemical emulsion as an adjuvantto radiation therapy in mice. Cancer Res. 44, 4285-4288, 1984].

Chemically, such a multifunctional delivery vehicle can be made offluorocarbon nanoparticles with chelators and peptides attached to theirsurfaces. The nanoparticles can be assembled in a modular fashion fromthe various components and can be formulated as oil-in-water (o/w)microemulsions. The chelators act as carriers for metallic radionuclides(e.g., ⁹⁰Y³⁺). The chelators can also carry other metallic ions, such asGd³⁺ for contrast-enhanced ¹H MRI. The peptides are for tumor targeting.The fluorocarbon nanoparticles themselves play three rolessimultaneously: ¹⁹F imaging agent, which derives from the high ¹⁹Fpayload of the nano-particles; pO₂ probe, which derives from thedependence of the relaxation behavior of ¹⁹F on pO₂ (¹⁹F MR relaxometry)[Parhaml, P. & Fung, B. M. Fluorine-19 relaxation study of perfluorochemicals as oxygen carriers. J. Phys. Chem. 87, 1928-1931, 1983]; O₂carrier, which derives from the high solubility of O₂ in fluorocarbonmicroemulsions [Riess, J. G. Understanding the fundamentals ofperfluorocarbons and perfluorocarbon emulsions relevant to in vivooxygen delivery. Art. Cells, Blood Subs., and Biotechnology, 33, 47-63,2005].

Note that since each nanoparticle carries both Gd³⁺ ions andfluorocarbons, it is a ¹H-¹⁹F dual nuclei MR imaging agent. While ¹Himaging is good at proving exquisite anatomical details due to the highintensity of the ¹H₂O signal, ¹⁹F imaging is ideal for drugconcentration determination as there is no detectable background ¹⁹Fsignal in the human body [Lanza, et al., Targeted antiproliferative drugdelivery to vascular smooth muscle cells with a magnetic resonanceimaging nanoparticle contrast agent. Circulation, 106, 2842-2847, 2002;Morawski, et al., Quantitative “magnetic resonance immunohistochemistry”with ligand-targeted 19F nanoparticles. Magn. Reson. Med. 52, 1255-1262,2004]. Note that ¹⁹F is the second most sensitive nucleus for MR imagingwith a sensitivity of 83% of that of ¹H. Hence a ¹H-¹⁹F dual nuclei MRimaging agent can simultaneously visualize tumor via the ¹H mode andtrace the drug and quantify local drug concentration via the ¹⁹F mode.Such local drug concentration determination will help with calculatingthe radiation dose delivered to the tumor site, a very importantparameter in radiotherapy (microdosimetry) [Bacher, K. & Thierens, H. M.Accurate dosimetry: an essential step towards good clinical practice innuclear medicine. Nucl. Med. Commun. 26, 581-586, 2005; Bardiès, M. &Pihet, P. Dosimetry and microdosimetry of targeted radiotherapy. CurrentPharmaceutical Design, 6, 1469-1502, 2000]. Local radiation dosimetrytypically requires experimental calibration between radiation dose and¹⁹F signal intensity.

¹⁹F MR relaxometry is a well-established non-invasive method for pO₂determination and is generally the only NMR technique which candetermine the absolute value of pO₂ [Swartz, H. M. & Dunn, J. F.Measurements of oxygen in tissues: overview and perspectives on methods.Adv. Experi. Med. Biol. 530, 1-12, 2003; Grucker, D. Oxymetry bymagnetic resonance: applications to animal biology and medicine. Prog.Nucl. Magn. Reson. Spect. 36, 241-270, 2000; Zhao, D., Jiang, L. &Mason, R. P. Measuring changes in tumor oxygenation. Methods inEnzymology, 386, 378-418, 2004; Robinson, S. P. & Griffiths, J. R.Current issues in the utility of 19F nuclear magnetic resonancemethodologies for the assessment of tumor hypoxia. Phil. Trans. R. Soc.Lond. B 359, 987-996, 2004]. The basic principle behind ¹⁹F MR oximetryis that the spin-lattice relaxation rate constant (R₁) of fluorocarbonemulsions has a linear dependence on pO₂ [Parhaml, P. & Fung, B. M.Fluorine-19 relaxation study of perfluoro chemicals as oxygen carriers.J. Phys. Chem. 87, 1928-1931, 1983]. The significance of oximetry inradiotherapy lies in the fact that pO₂ correlates with the outcome ofradiotherapy and hence can help with patient selection and doseoptimization [Nordsmark, M., Overgaard, M. & Overgaard, J. Pretreatmentoxygenation predicts radiation response in advanced squamous cellcarcinoma of the head and neck. Radiother. Oncology, 41, 31-39, 1996;Nordsmark, M. & Overgaard, J. A confirmatory prognostic study onoxygenation status and loco-regional control in advanced head and necksquamous cell carcinoma treated by radiation therapy. Radiother. Oncol.57, 39-43, 2000; Fyles, et al., Oxygenation predicts radiation responseand survival in patients with cervix cancer. Radiother. Oncolgy, 48,149-156, 1998; Brizel, D. M., Dodge, R. K., Clough, R. W. & Dewhirst, M.W. Oxygenation of head and neck cancer: changes during radiotherapy andimpact on treatment outcome. Radiother. Oncology, 53, 113-117, 1999;O'Hara, J. A., Goda, F., Demidenko, E. & Swartz, H. M. Effect onregrowth delay in a murine tumor of scheduling spit dose radiation basedon direct pO2 measurements by EPR oximetry. Radia. Res. 150, 549-556,1998; O'Hara, et al., Response to radioimmunotherapy correlates withtumor pO2 measured by EPR oximetry in human tumor xenografts. Radia.Res. 155, 466-473, 2001; Bussink, J., Kaanders, J. H. A. & van derKogel, A. J. Clinical outcome and tumor microenvironmental effects ofaccelerated radiotherapy with carbogen and nicotinamide. ActaOncologica, 38, 875-882, 1999; Kaanders, J. H. A. Bussink, J. & van derKogel, A. J. Clinical studies of hypoxia modification in radiotherapy.Seminars in Radiat. Oncol. 14, 233-240, 2004; Al-Hallaq, et al., MRImeasurements correctly predict the relative effects of tumor oxygenationagents on hypoxic fraction in rodent BA1112 tumors. Int. J. Radiat.Oncology Biol. Phys. 47, 481-488, 2000].

It has been demonstrated in both preclinical and clinical studies thatoxygen breathing (in the form of carbogen, 95% O₂ and 5% CO₂) inconjunction with fluorocarbon microemulsions can alleviate tumor hypoxiaand sensitize tumors toward radiation [Rockwell, S., Use of aperfluorochemical emulsion to improve oxygenation in a solid tumor. Int.J. Radiation Oncology Biol. Phys. 11, 97-103, 1985; Teicher, B. A. &Rose, C. M. Oxygen-carrying perfluorochemical emulsion as an adjuvant toradiation therapy in mice. Cancer Res. 44, 4285-4288, 1984; Al-Hallaq,et al., MRI measurements correctly predict the relative effects of tumoroxygenation agents on hypoxic fraction in rodent BA1112 tumors. Int. J.Radiat. Oncology Biol. Phys. 47, 481-488, 2000; Teicher, B. A. & Rose,C. M. Perfluorochemical emulsions can increase tumor radiosensitivity.Science, 223, 934-936, 1984; Rose, C., Lustig, R., McIntosh, N. &Teicher, B. A clinical trial of Fluosol DA 20% in advanced cellcarcinoma of the head and neck. Int. J. Radiat. Oncology Biol. Phys. 12,1325-1327, 1986; Koch, et al., Radiosensitization of hypoxic tumor cellsby dodecafluoropentane: A gas-phase perfluorocarbon emulsion. CancerRes. 62, 3626-3629, 2002]. Radiosensitization of tumor cells by O₂ isparticularly effective for high-energy β-emitters [Kassis, A. I. &Adelstein, S. J. Radiobiologic principles in radionuclide therapy. J.Nucl. Med. 46 (Suppl. 1) 4S-12S, 2005], such as ⁹⁰Y³⁺, the radionuclideused in Zevalin® and OctreoTher®.

A multifunctional delivery vehicle with octreotide as the targetingpeptide can be provided. Octreotide is the targeting peptide used inOctreoTher® [Smith, M. C., Liu, J., Chen, T., Schran, H., Yeh, C.-M.,Jamar, F., Valkema, R., Bakker, W., Kvols, L., Krenning, E. & Pauwels,S. OctreoTher™: ongoing early clinical development of asomatostatin-receptor-targeted radionuclide antineoplastic therapy.].This delivery vehicle can have application in targeted radiotherapy ofneuroendocrine tumors.

6. Fluorocarbon Nanoparticles in Biomedicine

Fluorocarbon nanoparticles, in the form of microemulsions, have a longhistory in biomedicine. Non-targeted fluorocarbon nanoparticles havebeen explored as blood substitutes [Riess, J. G. Understanding thefundamentals of perfluorocarbons and perfluorocarbon emulsions relevantto in vivo oxygen delivery. Art. Cells, Blood Subs., and Biotechnology,33, 47-63, 2005] and ultrasound contrast agents [Klibanov, A. L.Ligand-carrying gas-filled microbubbles: Ultrasound contrast agents fortargeted molecular imaging. Bioconjugate Chem. 16, 9-17, 2005]. In thefield of oncology, fluorocarbon nanoparticles have been used for tumorvisualization via ¹⁹F MR imaging [Longmaid, et al., In vivo 19F NMRimaging of liver, tumor, and abscess in rats. Invest. Radiol. 20,141-145, 1985; Ratner, et al., Detection of tumors with 19F magneticresonance imaging. Invest. Radiol. 23, 361-364, 1988; Mason, R. P.,Antich, P. P., Babcock, E. E., Gerberich, J. L. & Nunnally, R. L.Perfluorocarbon imaging in vivo: A ¹⁹F MRI study in tumor-bearing mice.Magn. Reson. Imag. 7, 475-485, 1989; Huang, M. Q., Basse, P. H., Yang,Q., Horner, J. A., Hichens, T. K. & Ho, C. MRI detection of tumor inmouse lung using partial liquid ventilation with aperfluorocarbon-in-water emulsion. Magn. Reson. Imag. 22, 645-652,2004], tumor oximetry via ¹⁹F MR relaxometry [Zhao, D., Jiang, L. &Mason, R. P. Measuring changes in tumor oxygenation. Methods inEnzymology, 386, 378-418, 2004; Robinson, S. P. & Griffiths, J. R.Current issues in the utility of 19F nuclear magnetic resonancemethodologies for the assessment of tumor hypoxia. Phil. Trans. R. Soc.Lond. B 359, 987-996, 2004; Mason, R. P., et al., Regional tumor oxygentension: Fluorine echo planar imaging of hexafluorobenzene revealsheterogeneity of dynamics. Int. J. Radiat. Oncology Biol. Phys. 42,747-750, 1998; van der Sanden, et al., Characterization and validationof non-invasive oxygen tension measurements in human glioma xenograftsby ¹⁹F-MR relaxometry. Int. J. Radiat. Oncology Biol. Phys. 44, 649-658,1999] and tumor radiosensitization via O₂ delivery [Rockwell, S. Use ofa perfluorochemical emulsion to improve oxygenation in a solid tumor.Int. J. Radiation Oncology Biol. Phys. 11, 97-103, 1985; Teicher, B. A.& Rose, C. M. Oxygen-carrying perfluorochemical emulsion as an adjuvantto radiation therapy in mice. Cancer Res. 44, 4285-4288, 1984;Al-Hallaq, H. A., Zamora, M., Fish, B. L., Farrell, A., Moulder, J. E. &Karczmar, G. S. MRI measurements correctly predict the relative effectsof tumor oxygenation agents on hypoxic fraction in rodent BA1112 tumors.Int. J. Radiat. Oncology Biol. Phys. 47, 481-488, 2000; Teicher, B. A. &Rose, C. M. Perfluorochemical emulsions can increase tumorradiosensitivity. Science, 223, 934-936, 1984; Rose, C., Lustig, R.,McIntosh, N. & Teicher, B. A clinical trial of Fluosol DA 20% inadvanced cell carcinoma of the head and neck. Int. J. Radiat. OncologyBiol. Phys. 12, 1325-1327, 1986; Koch, C. J., Oprysko, P. R., Shuman, A.L., Jenkins, W. T., Brandt, G. & Evans, S. M. Radiosensitization ofhypoxic tumor cells by dodecafluoropentane: A gas-phase perfluorocarbonemulsion. Cancer Res. 62, 3626-3629, 2002].

However, without targeting, the biodistribution of fluorocarbonnanoparticles is far from ideal. For example, in one study on ¹⁹F MRimaging of tumor in mice, it was found that the accumulation offluorocarbons in the spleen and liver is one to two orders of magnitudehigher than that in the tumor [Mason, R. P., Antich, P. P., Babcock, E.E., Gerberich, J. L. & Nunnally, R. L. Perfluorocarbon imaging in vivo:A 19F MRI study in tumor-bearing mice. Magn. Reson. Imag. 7, 475-485,1989]. Such a biodistribution profile is typically not acceptable fortherapeutic applications in humans.

The development of targeted fluorocarbon nanoparticles started in late1980's, using polyclonal antibodies [Shimizu, et al., Tumor imaging withanti-CEA antibody labeled 19F emulsion. Magn. Reson. Med. 5, 290-295,1987; Mishima, et al., In vivo F-19 shift imaging with FTPA andantibody-coupled FMIQ. J. Magn. Reson. Imag. 1, 705-709, 1991]. Morerecently, targeted nanoparticles based on monoclonal antibody or peptidemimetics have been developed for the detection of cardiovascularpathologies. Initially, these nanoparticles were developed as contrastagents for ultrasound imaging [Lanza, et al., A novel site-targetedultrasonic contrast agent with broad biomedical application.Circulation, 94, 3334-3340, 1996; Lanza, et al., Molecular imaging ofstretch-induced tissue factor expression in carotid arteries withintravascular ultrasound. Invest. Radiol. 35, 227-234, 2000; Lanza, G.M. & Wickline, S. A. Targeted ultrasonic contrast agents for molecularimaging and therapy. Progress Cardiovas. Disease, 44, 13-31, 2001].Later, their applications were extended into ¹H MR imaging [Anderson, etal., Magnetic resonance contrast enhancement of neovasculature withα_(v)β₃-targeted nanoparticles. Magn. Reson. Med. 44, 433-439, 2000;Flacke, et al., Novel M contrast agent for molecular imaging of fibrin.Circulation, 104, 1280-1285, 2001; Morawski, et al., Targetednanoparticles for quantitative imaging of spare molecular epitopes withMRI. Magn. Reson. Med. 51, 480-486, 2004; Schmieder, et al., MolecularMR imaging of melanoma angiogenesis with α_(v)β₃-targeted paramagneticnanoparticles. Magn. Reson. Med. 53, 621-627, 2005]. Most recently, thepotential of these targeted fluorocarbon nanoparticles as ¹⁹F MRcontrast agents was demonstrated through the ex vivo imaging of a humancarotid endarterectomy sample [Morawski, et al., Quantitative “magneticresonance immunohistochemistry” with ligand-targeted ¹⁹F nanoparticles.Magn. Reson. Med. 52, 1255-1262, 2004]. This pioneering work on targetedfluorocarbon nanoparticles demonstrated two points. First, targeting caneffectively improve the biodistribution. For example, in a preclinicalstudy conducted in dogs for blood clot detection, the contrast-to-noiseratio between the targeted clot and blood was ≈118±21 and thecontrast-to-noise ratio between targeted clot and control clot was131±37 [Flacke, et al., Novel MRI contrast agent for molecular imagingof fibrin. Circulation, 104, 1280-1285, 2001] (but no liver and spleenaccumulation data were presented). Second, targeted fluorocarbonnanoparticles can quantify the amount of drug delivered to apathological site via the ¹⁹F signal [Lanza, et al., Targetedantiproliferative drug delivery to vascular smooth muscle cells with amagnetic resonance imaging nanoparticle contrast agent. Circulation,106, 2842-2847, 2002; Morawski, et al., Quantitative “magnetic resonanceimmunohistochemistry” with ligand-targeted ¹⁹F nanoparticles. Magn.Reson. Med. 52, 1255-1262, 2004].

Current targeted fluorocarbon nanoparticles typically use egg yolkphospholipids (EYP) as emulsifying surfactants. This can lead to severalshortcomings. The first issue is stability. EYP are typically not verystable as they are susceptible to chemical modifications (e.g.,oxidation) [Riess, J. G. Oxygen carriers (“blood substitutes”)-raisond'etre, chemistry and some physiology. Chem. Rev. 101, 2797-2919, 2001;Tarara, T. E., Malinoff, S. H. & Pelura, T. J. Oxidative assessment ofphospholipid-stabilized perfluorocarbon-based blood substitutes. Art.Cells, Blood Subs., and Immob. Biotech. 22, 1287-1293, 1994]. This makestheir production, handling and storage problematic. Also, due to limitedinteraction between hydrocarbon and fluorocarbon compounds, the physicalstability of EYP-based fluorocarbon microemulsions is also not high. Thenanoparticles are prone to aggregation (Ostwald ripening) [Postel, M.,Riess, J. G. & Weers, J. G. Fluorocarbon emulsions—the stability issue.Art. Cells, Blood Subs., and Immob. Biotech. 22, 991-1005, 1994).

The second issue is derivatizability. The conjugation of phospholipidswith targeting molecules is not straightforward. It can require speciallinkers which themselves can present a problem. For instance, thebiotin-(strept)avidin linker system used in some targeted fluorocarbonnanoparticle [Lanza, et al., Targeted antiproliferative drug delivery tovascular smooth muscle cells with a magnetic resonance imagingnanoparticle contrast agent. Circulation, 106, 2842-2847, 2002;Morawski, et al., Quantitative “magnetic resonance immunohistochemistry”with ligand-targeted ¹⁹F nanoparticles. Magn. Reson. Med. 52, 1255-1262,2004; Lanza, et al., A novel site-targeted ultraosonic contrast agentwith broad biomedical application. Circulation, 94, 3334-3340, 1996;Flacke, et al., Novel MRI contrast agent for molecular imaging offibrin. Circulation, 104, 1280-1285, 2001; Morawski, et al., Targetednanoparticles for quantitative imaging of spare molecular epitopes withMRI. Magn. Reson. Med. 51, 480-486, 2004] can suffer from interferencefrom endogenous biotin and immunogenicity of (strept)avidin, asdemonstrated by studies on pretargeted radioimmunotherapy [Goldenberg,D. M. Targeted therapy of cancer with radiolabeled antibodies. J. Nucl.Med. 43, 693-713, 2002; Boerman, O. C., van Schaijk, F. G., Oyen, W. J.G. & Corstens, F. H. M. Pretargeted radioimmunotherapy of cancer:progress step by step. J. Nucl. Med. 44, 400-411, 2003; Knox, et al.,Phase II trial of yttrium-90-DOTA pretargeted by NR-LU-10antibody/streptavidin in patients with metastatic colon cancer. Clin.Cancer Res. 6,406-414, 2000; Hamblett, K. J., Kegley, B. B., Hamlin, D.K., Chyan, M. K., Hyre, D. E., Press, O. W., Wilbur, D. S. & Stayton, P.S. A streptavidin-biotin system that minimizes blocking by endogenousbiotin. Bioconjugate Chem. 13, 588-598, 2002]. Also, conjugationproducts can be heterogeneous if the derivatization reaction involvesamine (—NH₂) or thiol (—SH) groups because the targeting peptide or mAbmight have several such groups [Lanza, et al., Molecular imaging ofstretch-induced tissue factor expression in carotid arteries withintravascular ultrasound. Invest. Radiol. 35, 227-234, 2000]. Mostcritically, however, is that derivatization has very limited options,caused primarily by intrinsic limitations of phospholipid chemistry. Forexample, peptide-targeted fluorocarbon nanoparticles have not beendemonstrated previously. Peptide mimetic-conjugated nanoparticles,however, have been developed [Schmieder, et al., Molecular MR imaging ofmelanoma angiogenesis with α_(v)β₃-targeted paramagnetic nanoparticles.Magn. Reson. Med. 53, 621-627, 2005; Winter, et al., Molecular imagingof angiogenesis in nascent Vx-2 rabbit tumor using a novelα_(v)β₃-targeted nanoparticle and 1.5 tesla magnetic resonance imaging.Cancer Res. 63, 5838-5843, 2003]. The conjugation involves a thiolategroup which is not present in regular peptides.

The third issue is heterogeneity. There are several factors contributingto heterogeneity. The first factor is molecular diffusion andaggregation which lead to heterogeneity in nanoparticle size [Postel,M., Riess, J. G. & Weers, J. G. Fluorocarbon emulsions—the stabilityissue. Art. Cells, Blood Subs., and Immob. Biotech. 22, 991-1005, 1994].This issue is intrinsic to emulsion formulation. However, it is furtherconfounded by the use of EYP as the emulsifier. This is because EYP isnot a pure compound. Rather, it is a class of phospholipids, includingminor components such as phosphatedylinositol, phosphatidic acids,lysophosphatidylethanolamine, and fatty acids [Riess, J. G. Oxygencarriers (“blood substitutes”)-raison d'etre, chemistry and somephysiology. Chem. Rev. 101, 2797-2919, 2001]. Such minor components canundergo chemical modification (e.g., oxidation) during production. Theycan also participate in the conjugation reaction with targetingmolecules. Both processes can contribute to nanoparticle heterogeneity.This issue can be worsened if the composition of these minor componentsvaries from batch to batch. As a result, the size distribution ofcurrent targeted fluorocarbon nanoparticles can be very broad [Lanza, etal., Molecular imaging of stretch-induced tissue factor expression incarotid arteries with intravascular ultrasound. Invest. Radiol. 35,227-234, 2000; Winter, et al., Molecular imaging of angiogenesis innascent Vx-2 rabbit tumor using a novel α_(v)β₃-targeted nanoparticleand 1.5 tesla magnetic resonance imaging. Cancer Res. 63, 5838-5843,2003; Winter, et al., Improved molecular imaging contrast agent fordetection of human thrombus. Magn. Reson. Med. 50, 411-416, 2003]. Forexample, in one case of biotinylated fluorocarbon nanoparticles, thesize of the nanoparticles ranges from 50 nm to 1000 m with the averagearound 250 nm [Winter, et al., Improved molecular imaging contrast agentfor detection of human thrombus. Magn. Reson. Med. 50, 411-416, 2003]. Aconsequence of such broad size distribution is that the ¹⁹F signal isalso broad [Morawski, et al., Quantitative “magnetic resonanceimmunohistochemistry” with ligand-targeted ¹⁹F nanoparticles. Magn.Reson. Med. 52, 1255-1262, 2004]. Broad size distribution can make thebiodistribution of the nanoparticles difficult to control and may lowertherapeutic efficacy while broad ¹⁹F signal lowers the sensitivity of¹⁹F MRI.

7. Engineering of Targeted Fluorocarbon Nanoparticles

At the molecular level, EYP can be replaced by synthetic fluorinatedsurfactants (F-surfactants). This avoids numerous problems associatedwith EYP. Chemically, F-surfactants are very inert molecules, leading togreater chemical stability. Also, due to preferentialfluorocarbon-fluorocarbon interactions [Curran, D. & Lee, Z. Fluoroustechniques for the synthesis and separation of organic molecules. GreenChem. G3-G7, 2001], interactions between the outer shell (made ofF-surfactants) and inner core (made of F-oils) of the nanoparticles canbe much enhanced, leading to greater physical stability. Further, assynthetic molecules, minor components and batch-to-batch variations arenegligible. All these features should increase the stability whiledecrease the heterogeneity of fluorocarbon nanoparticles. Indeed,previous studies have shown that when EYP is replaced by F-surfactants,the stability of fluorocarbon microemulsions significantly increases[Riess, J. G. Oxygen carriers (“blood substitutes”)-raison d'etre,chemistry and some physiology. Chem. Rev. 101, 2797-2919, 2001; Postel,M., Riess, J. G. & Weers, J. G. Fluorocarbon emulsions—the stabilityissue. Art. Cells, Blood Subs., and Immob. Biotech. 22, 991-1005, 1994].However, new F-surfactants and F-oils, in particular the disclosedcompounds, can be used instead of commercially availablefluorochemicals.

Based on the molecular theories of the liquid state [Chandler, D.Structures of molecular liquids. Annu. Rev. Phys. Chem. 29, 441-471,1978], without wishing to be bound by theory, it is believed that, ifthe shape of the F-surfactant matches the shape of the F-oil, thestability and the heterogeneity of the nanoparticles are enhanced andreduced, respectively. The reason is that the “cavity” vacated by anF-oil molecule can be filled in snuggly by an F-surfactant molecule andvice versa.

To solve the issue of peptide conjugation, F-surfactants which can bedirectly conjugated to the N-terminus of peptides during solid-phasesynthesis can be synthesized. This simplifies the derivatization processand results in only one conjugation product, hence reducingheterogeneity. This method has general applicability toward thesolid-phase synthesis of any targeting molecules.

At the formulation level, a modular approach toward nanoparticleassembly is used. Specifically, an array of F-surfactants and F-oils canbe synthesized. The length and the charge of the hydrophilic moiety ofthe F-surfactants can be varied systematically. Further, someF-surfactants can be conjugated to a chelator (for carrying metallicradio-nuclides and/or stable metallic ions) while some can be conjugatedto a peptide (for tumor targeting). The F-oils can have varioushydrocarbon tails to modulate its lipophilicity, which is an importantconsideration in fluorocarbon nanoparticle formulation [Riess, J. G.Oxygen carriers (“blood substitutes”)-raison d'etre, chemistry and somephysiology. Chem. Rev. 101, 2797-2919, 2001]. Purified modules can bemixed together for nanoparticle constitution.

Modular nanoparticle assembly is central to achieving multifunctionalityas it allows one to integrate several functions into one deliveryvehicle through the incorporation of different modules. Anotherimportant consequence of modular assembly is that it allows us tomodulate the physicochemical properties of the nanoparticles (size,surface charge, chelator and peptide density, etc.) by mixing differentmodules at different molar ratios. Without wishing to be bound bytheory, it is believed that, by modulating physicochemical properties ofthe nanoparticles, their in vivo pharmacokinetics are modulated.

8. Targeted Radiotherapy

As shown in FIG. 2, a multifunctional delivery vehicle can be used inthree stages of targeted radiotherapy: pre-therapy planning,radiotherapy stage and post-therapy assessment.

a. Stage 1

At the pre-therapy planning stage (Stage 1), the delivery vehicle canhelp with two things. First, it can help with patient selection. This isaccomplished by measuring local pO₂ via ¹⁹F MR relaxometry. Once thepatient is selected, it can help with individualizing treatment plans byobtaining patient-specific pharmacokinetic information. This isaccomplished by tracing the drug surrogate via ¹⁹F MR and visualizingthe tumor via ¹H MR imaging. The aim is to determine whether tumortargeting is achieved and whether the overall biodistribution of thedrug is acceptable. The feasibility of ¹⁹F MRI to trace fluorocarbonnanoparticles or fluorinated drugs in vivo has been demonstrated byseveral previous studies [Meyer, K. L., Carvlin, M. J., Mukherji, B.,Sloviter, H. A. & Joseph, P. M. Fluorinated blood substitute retentionin the rat measured by fluorine-19 magnetic resonance imaging. Invest.Radiol. 27, 620-627, 1992; Kimura, A., Narazaki, M., Kanazawa, Y. &Fujiwara, H. ¹⁹F magnetic resonance imaging of perfluorooctanoic acidencapsulated in liposome for biodistribution measurement. Magn. Reson.Imag. 22, 855-860, 2004; Schlemmer, H. P., Becker, M., Bachert, P.,Dietz, A., Rudat, V., Vanselow, B., Wollensack, P., Zuna, I., Knopp, M.V., Weidauer, H., Wannenrmacher, M. & van Kaick, G. Alterations ofintratumoral pharmacokinetics of 5-fluorouracil in head and neckcarcinoma during simultaneous radiochemotherapy. Cancer Res. 59,2363-2369, 1999; Brix, G., Bellemann, M. E., Haberkom, U., Gerlach, L. &Lorenz, W. J. Assessment of the biodistribution and metabolism of5-fluorouracil as monitored by ¹⁸F PET and ¹⁹F MRI: a comparative animalstudy. Nucl. Med. Biol. 23, 897-906, 1996; Brix, G., Schlicker, A.,Mier, W., Peschke, P. & Bellemann, M. E. Biodistribution andphamacokinetics of the ¹⁹F-labeled radiosensitizer 3-aminobenzamide:assessment by ¹⁹F MR imaging. Magn. Reson. Imag. 23, 967-976, 2005].

At the pre-therapy planning stage, the cold surrogate can be given at adose that will saturate the receptor sites in normal tissues but not intumor tissues. This protects the normal tissues from radiation damagebut still leaves the tumor tissue vulnerable. Indeed, it is a standardpractice in targeted radiotherapy to first give a cold dose of drugsurrogate to protect the normal tissues from radiation [Drug LabelInformation for Zevalin® and Bexxar®. Available at Drugs@FDA.].

Currently in targeted radiotherapy (Zevalin®, Bexxar® and OctreoTher®),pre-therapy imaging is achieved via nuclear imaging (γ-scintigraphy)[Drug Label Information for Zevalin® and Bexxar®. Available atDrugs@FDA.; Bushnell, et al., Evaluating the clinical effectiveness of90Y-SMT 487 in patients with neuroendocrine tumors. J. Nucl. Med. 44,1556-1560, 2003; Wahl, R. L. Tositumomab and 131I therapy innon-Hodgkin's lymphoma. J. Nucl. Med. 46 (Suppl. 1), 128S-140S, 2005;Otte, A., Herrmann, R., Heppeler, A., Behe, M., Jermann, E., Powell, P.,Maecke, H. R. & Muller, J. Yttrium-90 DOTATOC: first clinical results.Eur. J. Nucl. Med. 26, 1439-1447, 1999; Paganelli, et al.,Receptor-mediated radiotherapy with 90Y-DOTA-D-Phe1-Tyr3-octreotide.Eur. J. Nucl. Med. 28, 426-434, 2001]. This can have two shortcomings.First, nuclear imaging typically increases the radiation burden to bothpatient and health care providers. This extra radiation burden isworsened if multiple rounds of imaging are needed. In the presentapproach, nuclear imaging is replaced by MR imaging. Second, in the caseof Zevalin® and OctreoTher®, nuclear imaging is done via a surrogateradio-nuclide, ¹¹¹In³⁺, a γ-emitter [Drug Label Information for Zevalin®and Bexxar®. Available at Drugs@FDA.; Bushnell, et al., Evaluating theclinical effectiveness of 90Y-SMT 487 in patients with neuroendocrinetumors. J. Nucl. Med. 44, 1556-1560, 2003; Otte, A., Herrmann, R.,Heppeler, A., Behe, M., Jermann, E., Powell, P., Maecke, H. R. & Muller,J. Yttrium-90 DOTATOC: first clinical results. Eur. J. Nucl. Med. 26,1439-1447, 1999; Paganelli, et al., Receptor-mediated radiotherapy with90Y-DOTA-D-Phe1-Tyr3-octreotide. Eur. J. Nucl. Med. 28, 426-434, 2001].The reason is that ⁹⁰Y³⁺ is a pure β-emitter and hence not suited fornuclear imaging. The working assumption is that the ⁹⁰Y³⁺-based drug andthe ¹¹¹In³⁺-based surrogate have identical biodistribution. However, asa result of differences in their coordination chemistry [Liu, S.,Pietryka, J., Ellars, C. E. & Edwards, D. S. Comparison of yttrium andindium complexes of DOTA-BA and DOTA-MBA: models for 90Y- and111In-labeled DOTA-biomolecule conjugates. Bioconjugate Chem. 13,902-913, 2002], this assumption is not always valid as demonstrated byseveral in vivo studies [Canera, et al., Eur. J. Nucl. Med. 21, 640-646,1994; Lövqvist, et al., PET imaging of 86Y-labeled anti-Lewis Ymonoclonal antibodies in a nude mouse model: comparison between 86Y and111In radiolabels. J. Nucl. Med. 42, 1281-1287, 2001]. Hence, there isan element of uncertainty associated with this ¹¹¹In³⁺-based surrogateapproach for biodistribution evaluation. In the disclosed approach,⁸⁹Y³⁺ replaces ¹¹¹In³⁺ as the surrogate for ⁹⁰Y³⁺. Since ⁹⁰Y³⁺ and ⁸⁹Y³⁺are isotopes, they have identical chemistry and hence uncertainty inbiodistribution evaluation is minimized if not abolished completely.

b. Stage 2

At the therapy stage (Stage 2), the ¹H-¹⁹F MR imaging capacity of thedelivery vehicle allows the physician to monitor the radiotherapeuticdrug directly in real time. Such real time feedback makes it possible toadjust treatment plans immediately. Currently in targeted radiotherapy,direct observation of the radiotherapeutic drug is not possible because⁹⁰Y³⁺ is suitable for nuclear imaging.

Also, at the therapy stage, the delivery vehicle has the potential tomodify the pharmacokinetics and the pO₂ profiles of each patient. pO₂modification is achieved via the O₂ delivery capacity of fluorocarbonmicro-emulsions, as mentioned before. Modification of pharmacokineticscan be achieved by modifying the physicochemical properties of thenanoparticles. Without wishing to be bound by theory, it is believedthat the pharmacokinetics of the nanoparticles can be modulated bymodulating their physicochemical properties. A well-known example ofusing nanoparticles to alter drug pharmacokinetics is delivering drugsthrough the blood-brain barrier via nanoparticles [Koziara, J. M.,Lockman, P. R., Allen, D. D. & Mumper, R. J. In situ blood-brain barriertransport of nanoparticles. Pharm. Res. 20, 1772-1778, 2003; Kreuter, J.Nanoparticle systems for brain delivery of drugs. Adv. Drug. Del. Rev.47, 65-81, 2001].

c. Stage 3

During the post-therapy assessment stage (Stage 3), the delivery vehiclecan be used to visualize the residual tumor (via ¹H-¹⁹F MR imaging) andevaluate its hypoxic status (via ¹⁹F MR relaxometry). Such informationhelps to determine whether the previous round of therapy is effectiveand whether another round of therapy is needed. If another round oftherapy is needed, the post-therapy assessment stage of the previousround automatically becomes the pre-therapy planning stage of the nextround. In such multiple-round targeted radiotherapy, a critical factorin determining the timing of the next round of therapy is tumor pO₂[O'Hara, J. A., Goda, F., Demidenko, E. & Swartz, H. M. Effect onregrowth delay in a murine tumor of scheduling spit dose radiation basedon direct pO2 measurements by EPR oximetry. Radia. Res. 150, 549-556,1998; O'Hara, et al., Response to radioimmunotherapy correlates withtumor pO2 measured by EPR oximetry in human tumor xenografts. Radia.Res. 155, 466-473, 2001]. Hence, pO₂ measurement plays an important rolein the delivery of multi-rounds of targeted radiotherapy.

All the aforementioned functions of the delivery vehicle can be furtherextended. In terms of imaging, aside from carrying ⁹⁰Y³⁺ and Gd³⁺, thechelators can also carry radionuclides for nuclear imaging (e.g., ¹¹¹Infor γ-scintigraphy and ⁸⁶Y for positron emission tomography). Inaddition to ¹⁹F MR imaging, fluorocarbon nanoparticles can be used forultrasound imaging. For example, Optison® is an FDA-approved ultrasoundcontrast agent based on fluorocarbon microemulsions [Klibanov, A. L.Ligand-carrying gas-filled microbubbles: Ultrasound contrast agents fortargeted molecular imaging. Bioconjugate Chem. 16, 9-17, 2005]. Inaddition to pO₂ measurement, ¹⁹F relaxometry also has the potential fortemperature measurement [Berkowitz, B. A., Handa, J. T. & Wilson, C. A.Perfluorocarbon temperature measurements using 19F NMR. NMR inBiomedicine, 5, 65-68, 1992; Mason, R. P., Shukla, H. & Antich, P. P. Invivo oxygen tension and temperature: simultaneous determination using19F NMR spectroscopy of perfluorocarbon. Magn. Reson. Med. 29, 296-302,1993]. Hence, these fluorocarbon nanoparticles can be easily convertedunto multi-modality and multi-purpose imaging agents. In terms oftargeting, more than one targeting molecules can be conjugated tonanoparticle surface as a result of the disclosed modular assemblingapproach. Hence, the nanoparticles can be converted into multi-targetingdelivery vehicles. In terms of drug delivery, the nanoparticles cancarry chemo-therapeutic agents in addition to radionuclides (e.g.,encapsulation of paclitaxal). Similarly, the nanoparticles can alsocarry other radiotherapy adjuvant (e.g., nicotamide) or other hypoxiamarkers (e.g., fluorinated 2-nitro-imidazoles). Hence, the nanoparticlescan be converted into multimodal delivery vehicles for combinedradio-/chemo-therapy.

9. Nanoparticle Formulation

Fluorocarbon nanoparticle emulsions, using the synthesized F-oils andF-surfactants, can be prepared. FIG. 3 shows pictures of two suchformulations. In both cases, the starting materials are 200 mg of F-oil(R_(o)=-Me) and 400 mg of PBS. The two F-surfactants have the same endgroup (R_(s)═—OH). The lengths of the oxyethylene unit for the top row(labeled F27-Me) and the bottom row (labeled F27-8OH) surfactants are 4and 8, respectively. The F-surfactants were added to the vial graduallyand the numbers between the two rows indicate the amount of addedF-surfactant (in mg). (The color of the last 4 pictures in the bottomrow is distorted somewhat due to the contrast of the wall which does notappear in other picture.) From visual observation, it is clear thatdifferent surfactants lead to different emulsions (one clear and oneturbid), indicating that nanoparticle properties can be adjusted bymodifying structures of F-oils and F-surfactants.

FIG. 4 shows the ¹⁹F NMR spectra of fluorocarbon nanoparticles for onerepresentative spectrum. In the microemulsion sample, the ¹⁹F signalsare very sharp (peak width is about 0.1 ppm), but the ¹⁹F signals inpublished works are much broader with peak width on the order of 2-3ppm, about 20-30 time broader than the microemulsion sample. Withoutwishing to be bound by theory, it is believed that the broadness of the¹⁹F signals in the published work is caused by the broadness of the sizedistribution of the nanoparticles [Winter, et al., Improved molecularimaging contrast agent for detection of human thrombus. Magn. Reson.Med. 50, 411-416, 2003]. Regardless of origin, broad ¹⁹F NMR signalslead to reduced ¹⁹F MRI sensitivity. Hence, the present fluorocarbonnanoparticles have a much higher sensitivity for the same amount of ¹⁹Fcontent.

SAXS has been used to characterize the size of the nanoparticleformulation. FIG. 5 presents the SAXS analysis results. The nanoparticlewas made of an F-oil (300 mg, R_(o)=-Me), an F-surfactant (200 mg,R_(s)═—NH₃ ⁺, j=4) and PBS (600 mg). a. Scattering intensity I(Q) vs.amplitude of the scattering vector Q. The diffraction peak maximum at0.123 Å⁻¹ indicates long-range structural order on the order of 51 Å(=2π/0.123 Å). The origin of this diffraction peak is still underinvestigation. b. Pair-wise scattering distribution function P(r) vs.pair-wise distance. The radius of the nanoparticle is given by theposition of the negative trough, which is 33 Å. Hence, the averagediameter of the nanoparticles in this formulation is about 6.6 nm. Thelarge broad peak in b. indicates strong inter-particle interaction,consistent with the positively charged status of the F-surfactant.

The fluorocarbon nanoparticles can be self-assembled from fluorocarbonmodules with the inner core made of F-oils and the outer shell made ofF-surfactants. The design and selection of F-oils and F-surfactants takeboth chemical and biological needs into consideration.

a. Nanoparticle Design

Chemically, the design takes four things into consideration. First,signal for ¹⁹F MR imaging. Second, signal for ¹⁹F oximetry. Third,nanoparticle stability. Fourth, derivatizability with chelators andpeptides. Biologically, the design takes pharmacokinetics,biocompatibility, and toxicology into consideration. While the chemicalrequirements can be more easily translated into specifics of moleculardesign, biological requirements are typically met on a trial-and-errorbasis. Hence a strategy to meet the biological requirements is to makethe structures of the F-oils and F-surfactants amenable to systematicvariation. Nonetheless, some rudimentary measures, based on existingknowledge, are taken at the onset of molecular design to meet biologicalrequirements.

For ¹⁹F MR imaging, a single sharp peak is preferred. In one aspect,this requires that all the fluorine atoms in a fluorocarbon module areindistinguishable (i.e., symmetric molecular construct). Thisrequirement can rule out chain-like molecules such as perfluorooctylebromide because the ¹⁹F signal splits [Morawski, et al., Quantitative“magnetic resonance immunohistochemistry” with ligand-targeted 19Fnanoparticles. Magn. Reson. Med. 52, 1255-1262, 2004]. For ¹⁹F oximetry,—CF₃ is significantly more sensitive than —CF₂-sensitive toward pO₂[Mason, R. P. Non-invasive physiology: 19F NMR of perfluorocarbons. Art.Cell, Blood Subs., and Immob. Biotech. 22, 1141-1153, 1994]. Thisrequirement can rule out macrocyclic molecules such asperfluoro-15-crown-5 which contains cyclically symmetric —CF₂— groups.As a result, a series of spherically symmetric F-oil moleculescontaining multiple —CF₃ groups can be preferred.

As can be seen below, the F-oils are heavily or highly fluorinated butnot necessarily perfluorinated, i.e., they can have a hydrocarbonportion. This design feature is based on biocompatibility and toxicityconsiderations. It is known that in order for the F-oil molecules to beexcreted efficiently from the body, certain lipophilicity can be needed[Riess, J. G. Oxygen carriers (“blood substitutes”)-raison d'etre,chemistry and some physiology. Chem. Rev. 101, 2797-2919, 2001]. Thehydrocarbon portion of F-oils provides this lipophilicity. Further, theF-oils have a variable portion R_(o). This flexibility allows us tofurther adjust it lipophilicity. However, even though the F-oils are notper-fluorinated, their fluorine content (F %˜65%) is comparable to thatof perfluorocarbons used as blood substitutes (e.g., perfluorooctylbromide, or PFOB, F %=64.6%). Hence, their O₂-carrying capacities areexpected to be comparable to that of PFOB because such capacities dependprimarily on fluorine content [Riess, J. G. Oxygen carriers (“bloodsubstitutes”)-raison d'etre, chemistry and some physiology. Chem. Rev.101, 2797-2919, 2001].

Only three representative F-surfactant structures are shown here.F-surfactants with other types of terminal groups (e.g., R_(s)═—CNMe₂ ⁺)can also be prepared. Like the design of F-oils, the design ofF-surfactants also takes chemical and biological requirements intoconsideration. One salient feature is that the fluorocarbon portion ofF-surfactants can be identical to the fluorocarbon portion of F-oils.This is based on stability considerations. Without wishing to be boundby theory, it is believed that shape matching between the oil and thesurfactant can lead to enhanced microemulsion stability, based onmolecular theory of the liquid state [Chandler, D. Structures ofmolecular liquids. Annu. Rev. Phys. Chem. 29, 441-471, 1978]. In termsof derivatizability with peptides, one version of F-surfactants hascarboxylate (—COO⁻) as the end group. This allows it to be conjugated tothe N-terminus of a peptide during solid-phase synthesis without anyprotection group needed.

To meet biological requirements, the hydrocarbon tail of theF-surfactants has three variable portions. The stem of the hydrophilictail is made of an oligooxyethylene segment, (—OCH₂CH₂—)_(j). The lengthof this segment is j. When j is small (in the range from 2 to about 10),for each F-surfactant, j is a fixed number. In such a case, eachF-surfactant is a pure compound, not a mixture. A mixture of such purecompounds can be prepared simply by mixing these pure compounds. When jis large (>>10), then j does not have a fixed value as the surfactantwill be prepared from polymers. In such a case, each surfactant is amixture of multiple compounds. Fluorinated surfactants witholigooxyethylene segments as the hydrophilic moiety have been usedextensively for the emulsification of fluorocarbons [Mathis, G.,Leempoel, P., Ravey, J.-C., Selve, C. & Delpuech, J-J. A novel class ofnon-ionic microemulsions: fluorocarbons in aqueous solutions offluorinated poly(oxyethylene) surfactants. J. Am. Chem. Soc. 106,6162-6171, 1984; Cui, Z., Fountain, W., Clark, M., Jay, M. & Mumper, R.J. Novel enthanol-fluorocarbon microemulsions for topical geneticimmunization. Pharm. Res. 20, 16-23, 2003]. Such fluorinatedoligooxyethylene surfactants not only make fluorocarbon microemulsionsmore stable than EYP-based ones, but also make them more biocompatibleby reducing phagocytosis [Peng, C.-A. & Hsu, Y.-C. Fluoroalkylatedpolyethylene glycol as potential surfactant for perfluorocarbonemulsion. Art. Cells, Blood Subs., and Immob. Biotech. 29, 483-492,2001; Hsu, Y.-C. & Peng, C.-A. Diminution of phagocytosedperfluorocarbon emulsions using perfluoroalkylated polyethylene glycolsurfactant. Biochem. Biophys. Res. Comm. 283, 776-781, 2001]. In termsof length, a recent study on fluorinated oligooxyethylene surfactantsdemonstrated that the oxyethylene segment starts to curl around eachother and form micelles in water when the average segment length islonger than 8.7 [Li, Y., Chen, Z.-Q., Tian, J., Zhou, Y.-B., Chen, Z.-X.& Liu, Z.-J. Synthesis of novel type of hybrid fluorocarbon ionicsurfactants containing polyoxyethylene chain. J. Fluorine Chem. 126,888-891, 2005]. In certain aspects, the length of the oxyethylenesegment is limited to 8 units. The short length is made up by highdensity, i.e., every surfactant molecule has a (—OCH₂CH₂—)_(j) segment.

The second variable portion of the hydrocarbon tail of F-surfactants isthe end group following the (—OCH₂CH₂—)_(j) segment. To modulate thephysicochemical properties of the nanoparticles, terminal groups ofF-surfactants can be positively charged (e.g., —NH₃ ⁺), negativelycharged (e.g., —COO⁻) and neutral (e.g., —OH). The biological relevancyof this type of variations is that the pharmacokinetics of fluorocarbonmicroemulsions can be adjusted through the adjustment of thephysicochemical properties of the emulsifiers [Tsuda, Y., Yamanouchi,K., Okamoto, H., Yokoyama, K. & Heldebrant, C. Intravascular behavior ofa perfluorochemical emulsion. J. Pharmacobiodyn. 13, 165-171, 1990;Obraztov, V. V., Kabalnov, A. S., Makarov, K. N., Gross, U., Radeck, W.& Rudiger, S. On the interactions of perfluorochemical emulsions withliver microsomal membranes. J. Fluorine Chem. 63, 101-111, 1993; Klein,D. H., Jones, R. C., Keipert, P. E., Luena, G. A., Otto, S. & Weers, J.G. Intravascular behavior of perflubron emulsions. Collods & Surf. A:Physicochem. Engineering Aspects, 84, 89-95, 1994].

The third variable is dendron type of branching and this is bestrepresented by the generation number k. The number of terminal branchesis given by 2^(k) (i.e., for F-surfactants with one, two, four, eightand sixteen terminal branches, k=0, 1, 2, 3, and 4, respectively).

Finally, for metallic ion complexation and tumor targeting, the terminalgroups of certain surfactants can be conjugated to DOTA(F-surfactant-DOTA) or octreotide (F-surfactant-octreotide).

In one aspect, the compounds and/or compositions relate to ananoparticle comprising at least one of the disclosed compounds. Forexample, in the nanoparticle, R₄ can comprise a moiety having thestructure:

wherein R₇, R₈, and R₉ are, independently, H, CH₂CO₂H, or alkyl.

In a further aspect, the compound can comprise the structure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; and wherein R₇, R₈, and R₉ are,independently, H, CH₂CO₂H, or alkyl. In a further aspect, thenanoparticle can further comprise a metallic radionuclide (e.g, ⁹⁰Y³⁺)or non-ratioactive metallic ions (e.g., Gd³⁺). In a further aspect, R₄can comprise a moiety having the structure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; wherein A is O, S, or amino; and whereinR′ comprises a peptide. In a further aspect, the nanoparticle cancomprises a compound comprising the structure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; wherein A is O, S, or amino; and whereinR′ comprises a peptide.

The peptide can comprise octreotide and/or its various analogs (such as,but not restricted to, lanreotide, vapreotide, etc.). Other peptides(such as, but not restricted to, bombesin, vascoactive intestinalpeptide, cholecystokinin, substance, P, etc.) can also be used for thispurpose (L. Bodei, G. Paganelli & G. Mariani, Receptor radionuclidetherapy of tumors: a road from basic research to clinical applications.J. Nucl. Med. 47, 375-377, 2006). Further, anybody molecules (such as,but not restricted to, rituximab, ibritumomab, tositumomab, trastuzuamb)and other targeting molecules (e.g., siRNA) are also suitable.

b. Nanoparticle Formulation

The nanoparticles can be formulated as microemulsions. Prior tonanoparticle formulation, each module can be purified. TheF-surfactant-DOTA modules complex with Gd³⁺ first, using standardprotocols [Heppeler, et al., Radiometal-labelled macrocyclicchelator-derivatized somatostatin analogue with superb tumour-targetingproperties and potential for receptor-mediated internal radiotherapy.Chem. Eur. J. 5, 1974-1981, 1999]. The complexation product,F-surfactant-DOTA-Gd³⁺, can be further purified by HPLC and verified bymass spectrometry before use.

For nanoparticle formulation, mixture of the various modules can bepassed through a high-pressure microfluidic device (model M-110S fromMicrofluidics, Newton, Mass.), following published procures [Morawski,et al., Quantitative “magnetic resonance immunohistochemistry” withligand-targeted 19F nanoparticles. Magn. Reson. Med. 52, 1255-1262,2004; Lanza, et al., A novel site-targeted ultrasonic contrast agentwith broad biomedical application. Circulation, 94, 3334-3340, 1996].The size of the nanoparticle can be modulated by adjusting the ratio ofcharged vs. neutral surfactants during formulation as high surfacecharge can result in electrostatic repulsion and hence reduced size. Theintended radius of the nanoparticles is between 10-50 nm.

The dominant component of the outer shell of the nanoparticles can beneutral F-surfactants ending with the —OH group. Positively ornegatively charged F-surfactants can be added to the formulation asmodulators of physicochemical properties (size, surface charge,stability, etc.). Adjustment of the physicochemical properties of thenanoparticles can be achieved in a combinatorial fashion by combiningdifferent modules and by varying the molar ratios of different modules.For example, adding charged surfactants to fluorocarbon emulsions canenhance stability by preventing flocculation [Oleksiak, C. B., Habif, S.S. & Rosano, H. L. Flocculation of perfluorocarbon emulsions. ColloidsSurf. A Physicochem. Engineering Aspects, 84, 71-79, 1994]. In addition,data indicate that properties of the microemulsions can be modulated byvarying structures of F-surfactants (FIG. 3, page 40).

c. Nanoparticle Characterization

Basic features of the ¹⁹F, including chemical shift values, number ofpeaks, peak width, T₁ and T₂, can be characterized by NMR spectroscopy,using standard procedures. This serves as a quick screening in thatformulations with broad ¹⁹F peaks can be abandoned.

Nanoparticles can be visualized using freeze-fracture electronmicroscopy, using standard procedure [Postel, et al.,Fluorocarbon/lecithin emulsions: identification of EYP-coatedfluorocarbon droplets and fluorocarbon-empty vesicles by freeze-fractureelectron microscopy. Biochim. Biophys. Acta, 1086, 95-98, 1991]. Thiscan give a crude but quick estimation the size and heterogeneity ofnanoparticles. Formulations leading to much heterogeneity can beabandoned.

The average size of the nanoparticles can be determined by small angleX-ray scattering (SAXS) technique. X-rays are scattered by electrons,the scattering densities are simply the sum of the number of electronsper unit volume. Due to the high electron density of the fluorine atom,fluorinated nanoparticles are particularly suited to be characterized bySAXS [Riess, J. G. Fluorous micro- and nanophases with a biomedicalperspective. Tetrahedron, 58, 4113-4131, 2002].

Data can be measured using the SAXS instrument currently at theUniversity of Utah and described in [Heidorn, D. B. & Trewhella, J.Comparison of the crystal and solution structures of calmodulin andtroponin C. Biochemistry, 27, 909-915, 1988]. 20 μl of a fluorocarbonmicroemulsion sample can be loaded into a capillary of 1 mm diameter.The sample can be spun down in to the capillary and the scatteringmeasurements can be done at 25° C. (maintained by the sample waterbath). The x-ray scattering experiment typically takes minutes to hours.Hence, it measures the average sizes of the nanoparticles.

Data analysis follows established procedures on analyzing SAXS data ofmicro-emulsions [Wormuth, K. R. & Kaler, E. W. Microemulsifying polaroils. J. Phys. Chem. 93, 4855-4861, 1989; Pons, R., Ravey, J. C.,Sauvage, S., Stebe, M. J., Erra, P. & Solans, C. Structural studies ongel emulsions. Colloids & Surf. A: Physicochem. Engineering Aspects, 76,171-177, 1993]. Signal intensity in SAXS depends on the electron densitycontrast between a particle and its surrounding medium. Electron densityis calculated by dividing the number of electrons of molecule by its vander Waals volume (the van der Waals volume of a molecule is calculatedby adding up the van der Waals volumes of its constitutive groups,according to [Lepori, L. & Gianni, P. Partial molar volumes of ionic andnonionic organic solutes in water: a simple additivity scheme based onthe intrinsic volume approach. J. Solution Chem. 29, 405-447, 2000)).For the present nanoparticles, electron densities of the inner score(made of F-oils and the fluorocarbon head of F-surfactants) and theouter shell (made of oxyethylene segments) are 0.85e/Å³ and 0.57e/Å³,respectively. The electron density of water is 0.33e/Å³. As a result,the electron density contrast (calculated as the square of thedifference between the electron density of the component and that ofH₂O) between the fluorocarbon inner core and H₂O is 4.7 times greaterthan the electron density contrast between the oxyethylene outer shelland H₂O. This constitutes the basis for separately obtaining the radiusof the inner core and the radius of the entire nanoparticle.

Specifically, to obtain the radius of the fluorocarbon inner core, KClis added to PBS buffer to match the electron density of the oxyethyleneouter shell. This way, the outer shell makes no contribution toscattering, and the radius of the inner core, r_(core), is obtained. Toobtain the radius of the entire nanoparticle, no KCl would be added. Inthis case, both the inner core and the outer shell make contribution tothe scattering intensity and consequently one can obtain the radius ofthe entire nanoparticle, r_(NP).

From r_(core), the average volume of the fluorocarbon inner core,v_(core), can be calculated as:

$\begin{matrix}{v_{core} = {\frac{4\pi}{3}\left( r_{core} \right)^{3}}} & (1)\end{matrix}$

From r_(NP), the average volume of the entire nanoparticle, v_(NP), canbe calculated in a similar fashion. Such volume information can beuseful in determining nanoparticle concentration.

d. Nanoparticle Stability

In vitro stability of the microemulsion can be evaluated by followingthe average size of the nano-particles over time, using SAXS. This hasproven to be an effective method in monitoring fluorocarbon emulsionstability [Trevino, L., Solé-Violan, L., Daumur, P., Devallez, B.,Postel, M. & Riess, J. G. Molecular diffusion in concentratedfluorocarbon emulsions and its effect on emulsion stability. New J.Chem. 17, 275-278, 1993].

e. Nanoparticle Concentration

Nanoparticle concentration can be calculated using the followingequation [Morawski, et al., Quantitative “magnetic resonanceimmunohistochemistry” with ligand-targeted 19F nanoparticles. Magn.Reson. Med. 52, 1255-1262, 2004]:

$\begin{matrix}{\lbrack{NP}\rbrack = \frac{V_{core}}{v_{core} \cdot V_{E} \cdot N_{av}}} & (2)\end{matrix}$where v_(core) is the volume of the fluorocarbon inner core ofindividual nanoparticles (determined by SAXS), V_(core) is the totalvolume of the fluorocarbon phase, V_(E) is the volume of the emulsionand N_(av) is Avogadro's number. V_(core) is calculated as:V_(oil,add)+f_(F)·V_(surf,add), where V_(oil,add) and V_(surf,add) arevolumes of added F-oil and F-surfactant, respectively, and f_(F) is thevolume fraction of the fluorocarbon head of the F-surfactant. f_(F) canbe calculated from the van der Waals volumes of the constitutive groupsof the F-surfactant [Lepori, L. & Gianni, P. Partial molar volumes ofionic and nonionic organic solutes in water: a simple additivity schemebased on the intrinsic volume approach. J. Solution Chem. 29, 405-447,2000].

In the case that phase separation occurs, meaning only part of addedF-oil and F-surfactant are incorporated into the nanoparticles, thefluorocarbon concentration in each phase can be determined by NMRspectroscopy, using a known amount of CF₃-containing compound (e.g.,trifluoroethanol, CF₃CH₂OH) as an internal standard. From thisconcentration and the volume of each phase, the fraction of incorporatedFoil and F-surfactant can be calculated. Then, volumes of incorporatedFoil and F-surfactant, V_(oil,incorp.) and V_(surf,incorp),respectively, can be calculated. These volumes can then be used tocalculate V_(core) as: V_(oil,incorp.)+f_(F)·V_(surf,incorp.).

f. Nanoparticle Payload

The concentration of Gd³⁺ in an emulsion preparation can be determinedby inductively coupled plasma optical emission spectrometry (ICP-OES).This method has been used routinely in determining Gd³⁺ content incontrast agent samples [Zong, Y., Wang, X., Goodrich, K. C., Mohs, A.,Parker, D. & Lu, Z. R. Contrast enhanced tumor MRI with newbiodegradable macromolecular Gd(III) complexes in mice. Magn. Reson.Med. 53, 835-842, 2005]. Samples of known Gd³⁺ concentrations can beused as standards for calibration.

The concentration of octreotide in each microemulsion sample can bedetermined by UV absorption spectroscopy. Trp, Tyr and the disulfidebond together gives an extinction coefficient of 7110 M⁻1·cm⁻¹ at 280 nm[Gill, S. C. & von Hippel, P. H. Calculation of protein extinctioncoefficients from amino acid sequence data. Analyt. Biochem. 182,319-326, 1989]. Note that no other components of the nanoparticlesabsorb UV light around 280 nm. If needed, the sample can be diluted by7M guanidinium chloride solution to dissolve the nanoparticle. Ifuncertainty still arises, the peptide concentration can be furtherdetermined by amino acid analysis (in which the peptide is broken intoamino acids by concentrated HCl) and nitrogen analysis (in which thepeptide is digested completely by concentrated HClO₄ and the amount ofrelease ammonium is determined). For example, see [Yu, Y., Makhatadze,G. I., Pace, C. N. & Privalov, P. L. Energetics of ribonuclease T1structure. Biochemistry, 33, 3312-3319, 1994]. From the concentrationratio of Gd³⁺ vs. nanoparticle and that of octreotide vs. nanoparticle,the payload Gd³⁺ and octreotide payload per nanoparticle can bedetermined.

g. Nanoparticle Biodistribution

For preliminary biodistribution evaluation, the retention of thenanoparticle in the tumor and spleen and liver can be determined. Spleenand liver are selected because these are the organs where fluorocarbonsaccumulate through the reticuloendothelial system [Mason, R. P., Antich,P. P., Babcock, E. E., Gerberich, J. L. & Nunnally, R. L.Perfluorocarbon imaging in vivo: A 19F MRI study in tumor-bearing mice.Magn. Reson. Imag. 7, 475-485, 1989; Riess, J. G. Oxygen carriers(“blood substitutes”)-raison d'etre, chemistry and some physiology.Chem. Rev. 101, 2797-2919, 2001; Ratner, A. V., Hurd, R., Muller, H. H.,Bradley-Simpson, B., Pitts, W., Shibata, D., Sotak, C. & Young, S. W.19F magnetic resonance imaging of the reticuloendothelial system. Magn.Reson. Med. 5, 548-554, 1987]. In the R33 phase, more detailsbiodistribution studies in involving other tissues and organs (blood,kidneys, brain, lung, etc.) can be determined.

The biodistribution evaluation compares the tumor-to-organ ratios oftargeted versus non-targeted nanoparticles. Three differentnanoparticles and their non-targeted counterparts (hence, a total of sixsamples) can be evaluated. The three nanoparticles can have neutral,positively and negatively charged F-surfactants, respectively(R_(s)═—OH, —NH₃ ⁺ and COO⁻). In one aspect, the non-targetednanoparticles can be decorated with DOTA-Gd³⁺, but no peptide. In afurther aspect, a more rigorous control in which nanoparticles decoratedwith a non-targeting octreotide analog can be used.

h. Tumor Cell Lines and Animal Models

For biodistribution and imaging studies, the well characterized AR4-2Jpancreatic carcinoma cell line (commercially available from AmericanType Cell Culture, ATCC) [Viguerie, N., Tahiri-Jouti, N., Esteve, J. P.,Clerc, P., Logsdon, C., Svoboda, M., Susini, C., Vaysse, N. & Ribet, A.Functional somatostatin receptors on a rat pancreatic acinar cell line.Am. J. Physiol. 255, G113-G120, 1988] can be used. This cell line isselected because of its exclusive constitutive expression of type 2somatostatin receptor (sstr2) [Froidevaux, et al., Differentialregulation of somatostatin receptor type 2 (sst 2) expression in AR4-2Jtumor cells implanted into mice during octreotide treatment. Cancer Res.59, 3652-3657, 1999]. sstr2 is the receptor subtype most commonlyover-expressed by neuroendocrine tumors and is the targeted foroctreotide and its analogs [Kaltsas, G. A., Papadogias, D., Makras, P. &Grossman, A. B. Treatment of advanced neuroendocrine tumours withradiolabelled somatostatin analogues. Endocrine-Related Cancer, 12,683-699, 2005]. AR4-2J cell line is derived from Wistar rats.

This cell line is implanted subcutaneously in Lewis rats. Lewis rats aresuitable for this work because they are an inbred strain derived fromthe Wistar rat and therefore exhibit little genetic variability. Tumormodels using this cell line in Lewis rats are already established[Storch, et al., J. Nucl. Med. 46, 1561-1569, 2005].

10⁷ AR4-2J pancreatic tumor cells can be injected subcutaneously in6-week old Lewis rats [Storch, et al., J. Nucl. Med. 46, 1561-1569,2005]. Two weeks after tumor inoculation, the rats can be injected viathe tail vein with nanoparticles at a ¹⁹F dose of 50 mmole/kg (forrationale on dose selection, see the following section). As statedabove, a total of six samples can be tested. For each sample, 6 rats (3male/3 female) can be used. 3, 7 and 14 days after nanoparticleinjection, animals from each group can be sacrificed (see section F fordetails of animal protocols). Tumor implants, liver and spleen can becollected from each animal for biodistribution assays. The total numberof animals used is ca. 110.

A sample can be mixed with ultra pure water and homogenized at 9500 rpmfor several minutes until there was no visible solid tissue. Each samplecan be halved equally in so that the amount of nanoparticles in eachhomogenized sample can be determined by two independent methods. Thefirst one determines the amount of fluorocarbon content. The second onedetermines the amount of Gd³⁺.

To determine fluorocarbon content, a known amount trifluoroethanol(CF₃CH₂OH) can be added to the sample as internal reference. The samplecan then be loaded into an NMR tube and the ¹⁹F signal intensity can bedetermined by NMR spectroscopy. ¹⁹F signal intensity can be convertedinto fluorocarbon concentration using the internal reference as thestandard. This method has been used previously to determine thebiodistribution of fluorocarbon emulsions in animal models [Zarif, etal., Biodistribution of mixed fluorocarbon-hydrocarbon dowel moleculesused as stabilizers of fluorocarbon emulsions: a quantitative study byfluorine nuclear magnetic resonance (N). Pharm. Res. 11, 122-127, 1994;McGoron, et al., Art. Cells, Blood Subs., and Immob. Biotech. 22,1243-1250, 1994].

Gd³⁺ content in each sample can be determined by ICP-OES. This method isused routinely in the determination of Gd³⁺ retention in tissues [Zong,Y., Wang, X., Goodrich, K. C., Mohs, A., Parker, D. & Lu, Z. R. Contrastenhanced tumor MRI with new biodegradable macromolecular Gd(III)complexes in mice. Magn. Reson. Med. 53, 835-842, 2005].

Fluorocarbon and Gd³⁺ tissue retention can be represented by percentageof injected dose per organ. Fluorocarbon content and Gd³⁺ content ineach organ can be compared for consistency test, using the ¹⁹F-to-Gd³⁺ratio in the original microemulsion as the reference point.

An HPLC-MS-based method can be used as an alternative to ¹⁹F NMR[Hansen, K. J., Clemen, L. A., Ellefson, M. E. & Johnson, H. O.Compound-specific, quantitative characterization of organicfluorochemicals in biological matrices. Environ. Sci. Technol. 35,766-770, 2001]. This highly sensitive method has been used successfullyto determine the concentration of fluorocarbons in human and monkeyorgans and blood samples [Olsen, G. W., Hansen, K. J., Burris, J. M. &Mandel, J. H. Human donor liver and serum concentrations ofper-fluorooctanesulfonate and other perfluorochemicals. Environ. Sci.Technol. 37, 888-891, 2003; Butenhoff, J. L., Kennedy, G. L.,Hinderliter, P. M., Lieder, P. H., Jung, R., Hansen, K. J., Gornan, G.S., Noker, P. E. & Thomford, P. J. Pharmacokinetics ofperfluorooctanoate in cynomolgus monkeys. Toxicol. Sci. 82, 394-406,2004].

The dose of fluorocarbon nanoparticles can be based on fluorine contentfrom the F-oil molecules which form the inner core of nanoparticles.F-oils can be the dominant fluoro-carbon component of the nanoparticlesand ¹⁹F MRI signal intensity can be determined by contributions fromF-oils. Technically, the concentration of this particular ¹⁹Fconcentration can be determined by NMR spectroscopy, using a knownquantity of trifluoroethanol (CF₃CH₂OH) as the standard. From thisconcentration value, the ¹⁹F dose in terms of mmole/kg can bedetermined.

As for dose selection, Table 1 lists ¹⁹F doses of various fluorocarbonemulsions. ¹⁹F refers to all the fluorine content in a fluorocarbonmicroemulsion. Some of the fluorocarbons give a single ¹⁹F single (e.g.,perfluoro-15-crown-5) while others give multiple ¹⁹F signals (e.g.,perfluorooctyl bromide). The ¹⁹F dose range is from 25 to 598 mmole/kgbody weight. Consequently, 50 mmol/kg can be used as the ¹⁹F dosage.

TABLE 1 ¹⁹F doses Applications Dose and route Subjects I. Targetedfluorocarbon microemulsions for ¹⁹F tumor imagingperfluorotributylamine, C₁₂F₂₇N 150 mmole ¹⁹F/kg¹, i.v. mice (Schimizuet al., 1987) II. Non-targeted fluorocarbon microemulsions for ¹⁹F tumorimaging perfluorotributylamine, C₁₂F₂₇N 363 mmole ¹⁹F/kg, i.v. rats(Longmaid et al., 1985) perfluorooctyl bromide (PFOB), C₈F₁₇Br 340 mmole¹⁹F/kg, i.v. mice (Ratner et al., 1988) mixture of C₁₂F₂₇N, C₁₀F₁₈ andC₉F₂₁N 450 mmole ¹⁹F/kg¹, i.v mice (Mason et al., 1989) III.Non-targeted fluorocarbon microemulsions for ¹⁹F tumor oximetryperfluoro-15-crown-5, C₁₀O₅F₂₀ 180 mmole ¹⁹F/kg, i.v mice (McIntyre etal., 1999) perfluoro-15-crown-5, C₁₀O₅F₂₀ 145 mmole ¹⁹F/kg, i.v mice(van der Sanden et al., 1999 a, b) perfluoro-15-crown-5, C₁₀O₅F₂₀ 25mmole ¹⁹F/kg², i.v rats (Fan et al., 2002) PFOB, C₈F₁₇Br 284 mmole¹⁹F/kg/, i.v rats (Shukla et al., 1995) hexafluorobenzene, C₆F₆ 1-5mmole ¹⁹F/tumor, intra- rats (Zhao et al., 2001, 2004; Mason et al.,1998; tumor injection Hunjan et al., 1998) IV. Non-targeted fluorocarbonmicroemulsions for O₂ delivery to tumor Fluorosol D, C₁₀F₁₈ 150 mmole¹⁹F/kg¹, i.v mice (Teicher and Rose, 1984a) Fluorosol D, C₁₀F₁₈ 598mmole ¹⁹F/kg, i.v human (Rose et al., 1986) perfluoro-15-crown-5,C₁₀O₅F₂₀ 146 mmole ¹⁹F/kg, i.v rats (Al-Hallaq et al., 2000) PFOB,C₈F₁₇Br 102 mmole ¹⁹F/kg, retro- mice (Thomas et al., 1991; 1995)orbital sinus ¹Dose is converted from mmole/mouse to mmole/kg bodyweight assuming the weight of a mouse is 20 gram. ²Dose is convertedfrom mmole/rat to mmole/kg body weight assuming the weight of a rat is200 gram.

i. Average Radii of the Nanoparticles

For a nanoparticle with an average radius of 25 nm (referring to thefluorocarbon core diameter), v_(core) is 6.55×10⁻²³ m³. The molar volumeof such fluorocarbons is around 0.44 L/mole (calculated from the F-oilwith R_(o)=-Me density=1.75 g/mL, M.W.=775). Hence, the number of F-oilmolecule per nanoparticle is 9×10⁴. Since each F-oil has, in one aspect,27 identical fluorine atoms, the number of identical fluorine atoms is2.5×10⁶/particle. Similarly, for a nanoparticle with a radius of 50 nm,the number of fluorine atoms is 2.0×10⁷/particle. Hence, fornanoparticles in the range of 25 nm-50 nm, the number of fluorine atomsis 2.5×10⁶ to 2.0×10⁷ per particle. The density of sstr2 in the AR4-2Jcell line is ca. 10 nM [Froidevaux, et al., Differential regulation ofsomatostatin receptor type 2 (sst 2) expression in AR4-2J tumor cellsimplanted into mice during octreotide treatment. Cancer Res. 59,3652-3657, 1999]. Hence, in vivo ¹⁹F signal density in the tumor can bein the range of 25-200 mM, sufficient for ¹⁹F MR imaging [Morawski, etal., Quantitative “magnetic resonance immunohistochemistry” withligand-targeted ¹⁹F nanoparticles. Magn. Reson. Med. 52, 1255-1262,2004]. A previous study reported that at 1.5 T the minimum detectable¹⁹F level is 30 μM [Schlemmer, et al., Alterations of intratumoralpharmacokinetics of 5-fluorouracil in head and neck carcinoma duringsimultaneous radiochemotherapy. Cancer Res. 59, 2363-2369, 1999].

j. Pharmacokinetics of Fluorocarbon Nanoparticles

Thorough pharmacokinetic (PK) studies of fluorocarbon nanoparticles canbe conducted, using high tumor-to-organ ratio and rapid excretion(t_(1/2) ranging from hours to days, rather than weeks to months) asselection criteria to establish a quantitative relationship between thephysicochemical properties (size, charge, peptide and chelator payload,dose, etc.) and the PK profiles of the nanoparticles. Without wishing tobe bound by theory, it is believed that, by modulating physicochemicalproperties of the nanoparticles, their pharmacokinetics can bemodulated. A series of ¹H and ¹⁹F MR imaging experiments can beconducted to evaluate the various functions engineered for selectednanoparticles.

k. Fluorocarbon Nanoparticles as Drug Tracers

¹⁹F MR-based techniques can be implemented for estimation ofbiodistribution and tissue pharmacokinetics of the fluorocarbonnanoparticles. Accumulation of the nanoparticles in the tumor can beevaluated against liver and spleen, the major organs thatnon-specifically take up these particles. Without wishing to be bound bytheory, it is believed that the targeting moiety on the fluorocarbonnanoparticles enhances the accumulation of the nanoparticles in a tumorsignificantly compared to those nanoparticles that lacks the targetingcapability (i.e., carrying a non-targeting peptide). Signal enhancementin tumor can be increased, for example, ≧30% when the nanoparticlecarries a targeting peptide compared to a non-targeting one.

l. Fluorocarbon Nanoparticles as pO₂ Probes and O₂ Delivery Vehicles

Without wishing to be bound by theory, it is believed that the tumorperiphery can have significantly higher pO₂ than the tumor center;however, pO₂ of tumor center can be enhanced when the animal isbreathing carbogen (95% O₂ and 5% CO₂). Also without wishing to be boundby theory, it is believed that, comparing to carbogen breathing alone(i.e., without nanoparticles), the tumor pO2 can be significantlyincreased by both carbogen breathing and nanoparticles delivery. Highertumor-to-organ ratio and better excretion profile can be achieved bymodifying the chemistry of the nanoparticles.

m. Non-Targeting Analogs of Octreotide

To verify the effect of targeting on nanoparticle pharmacokinetics, anon-targeted counterpart of each targeted nanoparticle can be preparedfor comparison. A targeted nanoparticle is decorated with octreotide(sequence: DPhe1-c[Cys2-Phe3-DTrp-4-Lys5-Thr6-Cys7]-Thr8-NH₂), whichtargets sstr2 with high affinity and specificity. In a non-targetednanoparticle, octreotide can be replaced by its inactive analog in whichL-Thr6, located in the active site of octreotide, is replaced by D-Thr6(commercially available in protected form). A previous study hasdemonstrated such an L→D replacement completely abolishes the bindingaffinity of octreotide toward all somatostatin receptors [Reuter, J. K.,Mattem, R.-H., Zhang, L., Morgan, B., Hoyer, D. & Goodman, M. Synthesesand biological activities of sandostatin analogs containingstreochemical changes in positions 6 or 8. Biopolymers, 53, 497-505,2000]. Since these two peptides are otherwise identical, nanoparticlesdecorated with the L-Thr6→D-Thr6 analog of octreotide constitute aperfect control to verify the effect of octreotide targeting.

n. Pharmacokinetic Studies

Twenty different nanoparticles along with their non-targetedcounterparts, all at one dose: 50 mmole ¹⁹F/kg, can be screened for hightumor-to-organ ratios and rapid excretion (t_(1/2) in the range fromhours to days). PK of each nanoparticle each nanoparticle sample can beevaluated at three doses (the default choices are 10, 50 and 100 mmole¹⁹F/kg). Hence, again roughly 20 samples (6-7 different nanoparticles,each at three different doses) and their non-targeted counterparts canbe evaluated. The cell line and the animal model can be as discussedpreviously. Tumor implantation and nanoparticle doses can be asdiscussed previously. For each nanoparticle sample and for each dose,data can be collected in 6 rats (3 male/3 female). Animals can beanesthetized with ketamine/xylazine and injection of the microemulsionwill occur via tail vein. Anesthesia can be maintained with injection ofa combination of ketamine/xylazine. At 0, 0.25, 0.5, 1, 2, and 4 hoursafter administration, a 0.5 mL blood sample can be drawn from the tailvein for assessing the plasma washout profile of the fluorocarbonnanoparticles. To limit the amount of blood withdrawn from each animal,samples can be staggered so that each animal has samples taken at fourof the time points. This can allow collection of four samples perdesired time point. Following this sample, animals can be allowed torecover and returned to the vivarium where they can be housed inmetabolic cages. At 1, 3, 7, 14 and 28 days after injection, 6 rats canbe sacrificed and their organs harvested for biodistribution analysis.Tumor, liver, spleen, kidney, lung, brain, and heart can be separatelyhomogenized and assayed for nanoparticle concentration. Bloodconcentrations can also be measured at these time points. The functionof the metabolic cages is to collect urine and feces so that the amountof F-oils and F-surfactants, particularly the less volatileF-surfactants, excreted though the trine and feces can be estimated.

The concentration measurements from the plasma samples during the firstfour hours can be fit to a compartmental pharmacokinetic model using anaïve pooled method. This will allows investigation of how rapidly themicroemulsion is taken up into tissues. The tissue samples can beanalyzed using non-compartmental pharmacokinetic modeling methods. Bothapproaches use commercially available pharmacokinetic software(WinNonLin, Pharsight, Mountain View Calif.). The naïve pooledcompartmental pharmacokinetic model gives a general description of therate at which the emulsion leaves the plasma and is taken up in thetissues. A naïve pooled approach can be used because it allows all thesamples to be used in fitting one descriptive pharmacokinetic model withthe limited number of samples per animal [Ette, E. I. & Williams, P. J.Population pharmacokinetics II: estimation methods. Ann.Pharmacotherapy, 38, 1907-15, 2004]. Estimates of systemic and localC_(max), T_(max), AUC, Mean Residence Time, Clearance, and Distributioncan be made using WinNonLin. Accumulated drug in the organs can beassessed to identify relative efficacy in tissue targeting. Tissuetargeting efficacy can be assessed by comparing the AUC for the eachorgan of interest compared to the AUC in the tumor. This gives anindication of the relative amount of drug exposure each tissue receives[Norwich, K. H. Noncompartmental models of whole-body clearance oftracers: a review. Ann Biomed. Eng. 25, 421-39, 1997; Gillespie, W. R.Noncompartmental versus compartmental modelling in clinicalpharmacokinetics. Clin Pharmacokinetics, 20, 253-62, 1991]. Meanresidence time, which determines the average time that drug moleculesare present in the tissue, also provides a relative comparison oftargeting efficacy. The remaining parameters (maximumconcentration—C_(max), and time of maximum concentration—T_(max)) can beused for assessing whether the exposure of different organ systems arebioequivalent. This is another metric for comparing the efficacy of thetargeting capabilities of the microsomal constructs [Alvan, G.,Paintaud, G. & Wakelkamp, M. The efficiency concept in pharmacodynamics.Clin Pharmaco-kinetics, 36, 375-89, 1999].

Without wishing to be bound by theory, it is believed that theaccumulation of the nanoparticles in the tumor can be significantlyenhanced comparing to the same nanoparticles that lacks the targetingcapability (carrying the non-targeting octreotide analog). Since liverand spleen are major organs taking up fluorocarbons in a non-specificfashion [Ratner, A. V., Hurd, R., Muller, H. H., Bradley-Simpson, B.,Pitts, W., Shibata, D., Sotak, C. & Young, S. W. ¹⁹F magnetic resonanceimaging of the reticuloendothelial system. Magn. Reson. Med. 5, 548-554,1987], tumor retention of fluorocarbon nanoparticles with liver andspleen can be evaluated. The PK profile in the liver & spleen alsoprovides an assessment of potential radiation damage to normal tissuesin the therapy, when a radioactive moiety, ⁹⁰Y, is attached to thefluorocarbon nanoparticles.

o. Experimental Protocol

Accumulation of the nanoparticles in the tumor versus liver and spleenover a time course of 4 weeks can be compared over a group of ratsbearing subcutaneous pancreatic tumors. Three doses can be tested foreach type of nanoparticles: 10, 50 and 100 mmole ¹⁹F/kg. Long term (12w) retention of the nanoparticles in the liver and spleen can bedetermined on a group of normal rats; for each type of nanoparticles, adose of 50 mmole ¹⁹F/kg can be examined.

¹⁹F imaging can be performed on 4.7 T horizontal bore magnet using a¹H/¹⁹F dual tune volume coil. A spin echo pulse sequence can be used foracquisition of the ¹⁹F images. The nanoparticles give rise to two sharpyet closely spaced resonances in the ¹⁹F NMR spectrum; the peaks are0.25 ppm apart and represent the F-oil and F-surfactant component of thenanoparticle respectively (FIG. 4). Both peaks can be used for imaging.Transaxial (perpendicular to the long axis of the rat) ¹⁹F imagescovering the liver and spleen and tumor can be acquired and overlaidwith the H-1 MR images of the same orientation and slice thickness. Aphantom containing a known concentration of the same nanoparticles canbe placed beside the animal during imaging. ¹⁹F signals from liver &spleen and tumor can be integrated on each slice and summed up for thewhole organ; signal from the whole phantom will also be obtained; aratio of liver_(F19)/phantom_(F19) and tumor_(F19)/phantom_(F19) can beobtained. The ratio (i.e., normalized to phantom signal) is used as anindex for nanoparticle accumulation in the liver & spleen and tumorrespectively, and can be plotted over time. The accumulation in thetumor can be compared with that in the liver & spleen. The time courseof nanoparticle accumulation in the liver & spleen of normal rats (notumor) can be subjected to pharmacokinetic modeling [Wahl, R. L.Tositumomab and 131I therapy in non-Hodgkin's lymphoma. J. Nucl. Med. 46(Suppl. 1), 128S-140S, 2005], which provides a profile of nanoparticleretention and excretion.

p. Data Analysis

A group (n=7 for each nanoparticle dose and for a control nanoparticlethat carries a non-targeting peptide) of tumor bearing rats receives anintravenous injection of 50 mmole ¹⁹F/kg of the nanoparticle in salinewhen the tumor reaches about 10 mm×10 mm (measured from the twoorthogonal axis of the tumor). They can be subjected to MR studies, forexample, at day 1, 3, 7, 14, 21 and 28 days after injection of thenanoparticles. The tumor accumulation for various nanoparticles can becompared and can be compared to accumulation of nanoparticles without atargeting peptide; accumulation in the liver & spleen and tumor overtime will also be compared; paired t-test can be used for statisticalanalysis; P value ≦0.05 can be considered as statistically significant.

A group (e.g., n=7) of normal rats will receive i.v. injection of 50mmole ¹⁹F/kg nanoparticles and can be subjected to MR studies at 1, 3, 7days and followed by weekly scan up to 12 weeks after administration.Time course of liver & spleen retention of the nanoparticles can befitted to both single exponential and linear elimination models asdescribed previously [Meyer, K. L., Carvlin, M. J., Mukherji, B.,Sloviter, H. A. & Joseph, P. M. Fluorinated blood substitute retentionin the rat measured by fluorine-19 magnetic resonance imaging. Invest.Radiol. 27, 620-627, 1992] and half life (t_(1/2)) for the exponentialphase and linear phase of elimination can be obtained.

Owing to the targeting peptide on the nanoparticles and without wishingto be bound by theory, it is believed that that tumor retention of thenanoparticles can be significantly increased compared to nanoparticlesthat carry a non-targeting peptide (control particles). However,significant uptake in the liver & spleen is anticipated and can bemonitored by ¹⁹F MR; however, without wishing to be bound by theory, itis believed that that the nanoparticles can be cleared out from theliver & spleen sooner than the tumor. The amount of accumulation andtime of retention of the nanoparticles in the liver & spleen allowsestimation of the radiation damage introduced when the nanoparticlecarries ⁹⁰Y³⁺, a radioactive isotope. A more accurate estimate of ⁹⁰Ydistribution is to let the nanoparticle carry ⁸⁹Y³⁺ in addition to Gd³⁺.In addition, the potential toxicity of the fluorocarbon nanoparticles tothe liver & spleen can be examined by serum AGT level weekly and byhistological examination of the liver & spleen at 8^(th) week afteradministration of the nanoparticles (mice can be euthanized).

The Gd³⁺ moiety associated with the fluorocarbon nanoparticle provides atumor specific enhancement of ¹H signal on MR images. This property canbe extremely useful when tumor is located deep inside the body (e.g., apancreatic tumor) or when its size is small. The enhancement of tumorsignal can be compared before and 4, 24, 48 hours after administrationof the Gd³⁺ containing nanoparticles that contains either a targetingpeptide or a non-targeting peptide. Without wishing to be bound bytheory, it is believed that the signal enhancement in tumor can beincreased ≧30% when the nanoparticle carries a targeting peptidecompared to a non-targeting one.

Rats bearing subcutaneous tumors can be subjected to MR studies when thetumor reaches 10 mm×10 mm (measured from the two orthogonal axis of thetumor). A baseline scan (¹H and ¹⁹F imaging) can be performed before therat is injected i.v. with 10, 50 or 100 mmole ¹⁹F/kg nanoparticles; foreach dose, the particles that carry a non-targeting peptide can be usedas control; T₁-weighted and T₂-weighted ¹H images can be acquired alongwith ¹⁹F MR images at 4, 24, and 48 hours after administration of thenanoparticles.

¹⁹F images can be overlaid with T1W and T2W H-1 images; T1W imagesindicates the effect of paramagnetic reagent (in this case Gd³⁺) inwhich locations accumulating the Gd³⁺ can have higher signalintensities; on the other hand, T2W images provides information aboutcomponents of the tumor (necrotic, viable and edematous regions); byoverlaying ¹⁹F signal on ¹H images, important information regarding thelocation and extent of nanoparticle accumulation is gained. Signalintensities from tumor at pre- and post-contrast enhancement time pointscan be normalized to the intensity of phantom signal in the samefield-of-view before comparing with each other. This is to ensure thatany slight differences in receiver gain, magnetic homogeneity, etc., donot affect the quantification.

Without wishing to be bound by theory, it is believed that, with thetargeting moiety, the enhancement of tumor ¹H signal due to the presenceof Gd³⁺ can be ≧30% compared to non-targeted nanoparticles; due to longcirculation time of these particles, the accumulation will increase overtime so will the enhancement. By varying the payload of Gd³⁺ moieties,the enhancement is likely to be optimized to detect small size tumor.

¹⁹F MR relaxometry can be implemented for measurements of tumor pO₂ toevaluate O₂ delivery capacity of the fluorocarbon nanoparticles. TumorpO₂ can be an important parameter that determines the success ofradiotherapy because hypoxic tumor is resistant to radiation [Gray, L.H., Conger, A. D., Ebert, M., Hornsey, S. & Scott, O. C. Theconcentration of oxygen dissolved in tissues at the time of irradiationas a factor in radiotherapy. Br. J. Radiol. 26, 638-648, 1953; Hall; E.J., Radiobiology for the radiologist (4th edition). Lippincott,Philadelphia, 1994]. For a hypoxic tumor (pO2<10 mmHg), radiation dosethree times higher than that for an oxygenated tumor (pO2>30 mmhg) isneeded [Hall, E. J., Radiobiology for the radiologist (4th edition).Lippincott, Philadelphia, 1994]. Although polarographic needle oxygenelectrodes (Eppendorf Histograph) are widely used in the clinic, it isan invasive (destructive) technique and can only access to relativelysuperficial tumors [Nelson, et al., A noninvasive approach for assessingtumor hypoxia in xenografts: developing a urinary marker for hypoxia.Cancer Res. 65, 6151-6158, 2005]. A unique capability of ¹⁹F MR is toallow non-invasive estimation of tissue pO₂ by measuring itsspin-lattice relaxation time (T₁). By varying O₂ concentration in thephantom solution of fluorocarbon nanoparticles, a calibration curve (T₁vs pO₂) can be constructed at a specific temperature using the same MRcoil configuration and pulse sequence as for in vivo studies. Tumor pO₂will then be obtained from the calibration curve once its T₁ isdetermined by ¹⁹F MR. Non-invasive MR technique can be validated againstthe oxygen sensitive electrode polarographic measurements. Thefluorocarbon nanoparticles can then be evaluated for their sensitivityand accuracy for probing tumor pO₂.

Without wishing to be bound by theory, it is believed that tumorperiphery can have significantly higher pO₂ than the tumor center;however, pO₂ of a tumor center can be increased when an animal isallowed to breathe carbogen (95% O₂ and 5% CO₂). Also without wishing tobe bound by theory, it is believed that uptake of nanoparticles in thetumor can increase the tumor pO₂ when the animal breathes carbogenbeyond the level that is achieved by carbogen breathing alone.

As shown in Stage 2 of FIG. 2, carbogen breathing combined withfluorocarbon nanoparticle delivery provides a potential to increase thetumor pO₂ leading to sensitization of tumor to radiotherapy. Themechanism of carbogen breathing to increase the tumor oxygenation hasbeen studied extensively [Howe, F. A., Robinson, S. P., Rodrigues, L. M.& Griffiths, J. R. Flow and oxygenation dependent (FLOOD) contrast MRimaging to monitor the response of rat tumors to carbogen breathing.Magn. Reson. Imag. 17, 1307-1318, 1999] and carbogen breathing has beencombined with perfluorocarbon compounds for delivery of oxygen to thehypoxic tissues [Teicher, B. A. & Rose, C. M. Oxygen-carryingperfluorochemical emulsion as an adjuvant to radiation therapy in mice.Cancer Res. 44, 4285-4288, 1984; Al-Hallaq, et al., MRI measurementscorrectly predict the relative effects of tumor oxygenation agents onhypoxic fraction in rodent BA1112 tumors. Int. J. Radiat. Oncology Biol.Phys. 47, 481-488, 2000; Koch, et al., Radiosensitization of hypoxictumor cells by dodecafluoropentane: A gas-phase perfluorocarbonemulsion. Cancer Res. 62, 3626-3629, 2002].

q. Measurement of Tumor pO₂ by ¹⁹F-Relaxometry.

Rat pancreatic cancer (AR4-2J from American Type Tissue Culture, ATTC)over-expressing somatostatin receptor type 2 (sstr2) can be grownsubcutaneously in the hind limbs of rats. The animal can be subjected topO₂ measurements when tumor reaches to 10 mm×10 mm (measured from twoorthogonal axes of the tumor).

Calibration curves (T₁ versus pO₂) can be constructed on a phantomcontaining fluorocarbon nanoparticles in saline following establishedprocedures [Parhaml, P. & Fung, B. M. Fluorine-19 relaxation study ofperfluoro chemicals as oxygen carriers. J. Phys. Chem. 87, 1928-1931,1983; van der Sanden, et al., Characterization and validation ofnon-invasive oxygen tension measurements in human glioma xenografts by19F-MR relaxometry. Int. J. Radiat. Oncology Biol. Phys. 44, 649-658,1999; Mason, R. P., Shukla, H. & Antich, P. P. In vivo oxygen tensionand temperature: simultaneous determination using 19F NMR spectroscopyof perfluorocarbon. Magn. Reson. Med. 29, 296-302, 1993]. Duringconstruction of calibration curve, the pO2 in the solution is known andcontrolled while the corresponding T₁ value is measured by ¹⁹F MR. Note,same MR coil setting and pulse sequence can be used for phantom as wellas for in vivo studies; temperature of the phantom solution is monitoredby a thermister and is maintained to a specified value and a number ofcalibration curves corresponding to different temperatures can beconstructed.

Once the calibration curve is constructed, animals can be injectedintravenously (i.v.) the fluorocarbon nanoparticles; three doses, forexample, can be tested, 10, 50 and 100 mmole ¹⁹F/kg of body weight; ratscan be subjected to T₁ measurement. The ¹⁹F T₁ map can be generatedusing an imaging based multi-slice sequence developed previously [Zhou,et al., Simultaneous measurement of arterial input function and tumorpharmacokinetics in mice by dynamic contrast enhanced imaging: effectsof transcytolemmal water exchange. Magn. Reson. Med. 52, 248-257, 2004];on a T₁ map, each pixel intensity value represents its T₁ value and canbe converted to pO₂ value using the calibration curve. High resolutionT₂ weighted (T2W) H-1 spin echo images can be acquired; necrotic region(if any) can be identified on the T₂ weighted images; tumor can besegmented into peripheral and central region on the T₂ weighted imageswhich are overlaid on corresponding ¹⁹F images; regional pO₂ can beobtained by averaging the pO₂ of pixels in the specified region.

Immediately after ¹⁹F relaxometry, rats can be subjected to computerizedpolarographic needle electrode system (KIMOC 6650, Eppendorf) formeasurements of tumor pO₂. The polarographic needle has a diameter of300 μm with a sensitive membrane covered cathode of 17 μm, resulting ina hemispherical sensitive volume of 50 μm in diameter. In order to beable to compare pO₂ results from ¹⁹F relaxometry, polarographicmeasurements can be performed along two perpendicular tracks in thecorresponding tumor slices as used in the ¹⁹F relaxometry.

r. Nanoparticles as O₂ Delivery Vehicles

Without wishing to be bound by theory, it is believed that carbogenbreathing will enhance tumor pO₂ and the uptake of fluorocarbonnanoparticles in the tumor will further enhance tumor pO₂ beyond thatachieved by carbogen breathing alone. Rat pancreatic cancer (AR4-2J)overexpressing somatostatin receptor type II (SSTRII) can be grownsubcutaneously in the hind limbs of rats. When the tumor reaches to 10mm×10 mm (measured from two orthogonal axes of the tumor), tumor pO₂ canbe measured by Eppendorf needle electrodes with animal breathing air andthen carbogen; then the animal can be injected intravenously withfluorocarbon nanoparticles (10, 50 or 100 mmole ¹⁹F/kg); twenty-fourhours after nanoparticle administration, tumor pO₂ can be measured byEppendorf needle electrodes with animal breathing air and then carbogen.Note when the breathing gas is shifted from air to carbogen; astabilizing period of 5 min can be used before a pO₂ measurement startsand the rat remains inhaling carbogen during the measurement.

Three doses, with each dose having seven to ten (n=7-10) rats bearingsubcutaneous pancreatic cancer can be utilized. Tumor can be segmentedusing T2W images into peripheral (4 mm rim from the boundary of thetumor) and the region inside the rim can be counted as the centralregion. Regional pO₂ assessed by MR and by polarographic measurementscan be tabulated and compared using paired t-test. P value ≦0.05 can beconsidered as statistically significant.

The relationship between T₁ and pO₂ can be formulated in the equationbelow [Zhao, D., Jiang, L. & Mason, R. P. Measuring changes in tumoroxygenation. Methods in Enzymology, 386, 378-418, 2004]:R₁(≡1/T₁)=a+bpO₂  (3)

The ratio η=b/a has been proposed as a sensitivity index, therefore, ηvalues can be compared among various fluorocarbon nanoparticles toevaluate their sensitivity as a pO₂ probe. Seven to ten (n=7-10 for eachdose) rats bearing subcutaneous pancreatic tumors can be used. Regional(periphery and center) tumor pO₂ before and during carbogen breathingcan be compared using paired t-test. P value ≦0.05 can be considered asstatistically significant.

The ¹⁹F T₁ of fluorocarbon compound varies linearly with pO₂, and eachresonance is sensitive to pO2, temperature, and magnetic field, butimportantly, is essentially unresponsive to pH, CO₂, chargedparamagnetic ions, mixing with blood, or emulsification [Zhao, D.,Jiang, L. & Mason, R. P. Measuring changes in tumor oxygenation. Methodsin Enzymology, 386, 378-418, 2004]. Due to the temperature dependent ofT₁ of the nanoparticles, maintaining the temperature of the tumor thesame as the temperature under which the calibration curve is establishedis critical for accurate estimation of tumor pO₂. Two temperaturesensors can be used, one in the rectum (core temperature) and the otheron the surface of the subcutaneous tumor. Warm air can be directed tothe bore of the magnet to maintain the core temperature at 37±0.2° C.;once the core temperature is stabilized, the temperature on the tumorsurface can be used for the construction of the calibration curve.

It has been observed frequently by other investigators discrepanciesbetween ¹⁹F relaxometry and polarographic electrode based measurement oftumor pO₂: ¹⁹F relaxometry generally yields higher pO₂ values thanEppendorf electrodes (Robinson, S. P. & Griffiths, J. R. Current issuesin the utility of 19F nuclear magnetic resonance methodologies for theassessment of tumor hypoxia. Phil. Trans. R. Soc. Lond. B 359, 987-996,2004]. It has been suggested that this is primarily due to the ¹⁹Frelaxometry measurements are more weighted towards tumor periphery,where the blood vessels are more abundant and perfluorocarbon compoundsare carried in by perfusion; however, the needle electrodes do not havesuch bias. Without wishing to be bound by theory, it is believed thatsegmenting tumor into rim and central region and comparing the regionalmeasurement of pO₂ by the two methods decreases the discrepancies. Ifthe central region of the tumor is significantly necrotic, and thus hasvery limited perfusion leading to very low accumulation ofnanoparticles, only the tumor rim regions are compared. Without wishingto be bound by theory, it is believed that both methods are sufficientlysensitive to detect the difference in pO₂ pre- and post carbogenbreathing.

The amount of fluorocarbon nanoparticles accumulated in the tumordetermines whether the signal-to-noise ratio (S/N) of ¹⁹F signal issufficient for imaging, which yields a pixel-by-pixel map of pO₂ of thetumor. In case of insufficient S/N, unlocalized ¹⁹F spectroscopy isused, in which signal from whole tumor can be collected (using asolenoid coil) resulting in estimation of global pO₂ of the tumor. Insuch a case, the tumor periphery is not separated from tumor center;however, the spectroscopic method can be faster in measurements ofglobal tumor pO₂ and is typically sensitive enough to detect differencein pO₂ before and after carbogen breathing.

Based on results of in vivo imaging studies, the fluorous nanoparticlescan be modified and optimized. One goal of nanoparticle improvement canbe in pharmacokinetics, i.e., higher tumor-to-organ ratio and optimalexcretion profile (t½ in the range of hours to days, not minutes orweeks and months). The secondary goal can be in increasing imagingsensitivity of for drug tracing, tumor visualization and pO₂ oximetry.Modification of the nanoparticles can be conducted at both the molecularlevel and the formulation level, using the methods previously describedherein. For example, the nanoparticles can be made more hydrophiliceither at the molecular level incorporating more oxyethylene units toF-surfactants or at the formulation level by increasing the percentageof charged surfactants.

L. MICROBICIDAL FLUOROCARBON NANOEMULSIONS

Emulsions are ternary liquid systems made of oil, surfactant(emulsifier) and water. See, generally, [Hamouda, T., Myc, A., Donovan,B., Shih, A. Y., Reuter, J. D., Baker, J. R. A novel surfactantnanoemulsion with a unique non-irritant topical antimicrobial activityagainst bacteria, enveloped viruses and fungi. Microbiol. Res. 156, 1-7,2001; Myc, A., Vanhecke, T., Landers, J. J., Hamouda, T. & Baker, J. R.The fungicidal activity of novel nanoemulsion (X8W60PC) againstclinically important yeast and filamentous fungi. Mycopathologia, 155,195-201, 2001; Chepurnov, A. A., Bakulina, L. F., Dadaeva, A. A.,Ustinova, E. N., Chepurnova, T. S. & Baker, S. R. Inactivation of Ebolavirus with a surfactant nanoemulsion. Acta Tropica, 87, 315-320, 2003].In nanoemulsions, the size of oil droplets is on the order of several totens of nanometers, hence the name nanoemulsion. It has been found thatnanoemulsions have biocidal activities against a wide spectrum ofmicrobial organisms, including viruses (e.g., Ebola virus, herpessimplex type 1, influenza A, vaccinia virus, etc.), bacteria (e.g.,Bacillus cereus, Bacillus subtilis, Haemophilus influenza, Niesseriagonorrhoeae, Streptococcus pneumoniae, etc.) and fungi (e.g., Candidaalbicans, Microsporuin gypseum, Fusarium oxysporum, etc.). Withoutwishing to be bound by theory, the proposed mechanism of microbicialactivity is that the high surface energy of the nanoparticles causesthem to fuse with the outer membrane of the microbes, eventually causingthe outer membrane to burst, hence killing the microbes.

Engineered fluorocarbon nanoemulsions can have microbicidal activities.The nanoparticles in such an emulsion are made of highly fluorinatedoils and surfactants (F-oils and F-surfactants). The rationale is thatF-surfactants have high surface activities. Hence, nanoemulsionsformulated from F-oils and F-surfactants are more potent thanhydrocarbon nanoemulsions in terms of microbicial activities. Indeed,F-surfactants have been found in the past to have strong antibacterialand anti-HIV activities [Sawada, H., Ohashi, A., Baba, M., Kawase, T. &Hayakawa, Y. Synthesis and surfactant properties of fluoroalkylatedsulfonic acid oligomers as a new class of human immunodeficiency virusinhibitors. J. Fluorine Chem. 79, 149-155, 1996; Sawada, et al.,Synthesis of novel fluoroalkylated 4-vinylpyridinium chloride oligomersas functional materials possessing surfactant and biological properties.J. Fluorine Chem. 83, 125-131, 1997; Sun, J. Y., Li, J., Qiu, X.-L. &Qing, F.-L. Synthesis and structure-activity relationship (SAR) of novelperfluoroalkyl-containing quaternary ammonium salts. J. Fluorine Chem.126, 1425-1431, 2005].

There are at least three specific uses for such nanoemulsions; eachapplication takes advantage of unique properties of fluorocarbons. Thefirst application is for first-line prevention and treatment ofinfluenza and other respiratory viruses in the case of an outbreak. Forthis type application, the nanoemulsions can be formulated into aerosols(nasal sprays and inhalers). Note that fluorocarbons have traditionallybeen used in the formulation of aerosols [Lowe, K. C. Perfluorochemicalrespiratory gas carriers: benefits to cell culture systems. J. FluorineChem. 118, 19-26, 2002] and hence the present fluorocarbon-basednanoemulsions can have a distinct advantage in this regard. The secondapplication is for first-line prevention and treatment in the case ofbioterrorism attacks. In this case, the nanoemulsion can be formulatedinto either aerosols or sprays. Again, the weak intermolecularinteractions among fluorocarbon compounds make them well-suited for suchformulations. The third application is for preventing sexuallytransmitted diseases (STD). For this type of application, thenanoemulsions can be formulated as a gel. The weak intermolecularinteractions of fluorocarbons make them suitable for lubricant-basedanti-STD products (fluorocarbon materials can easily spread and are veryslippery [Lemal, D. M. Perspective on fluorocarbon chemistry. J. Org.Chem. 69, 1-11, 2004]). Further, due to the chemical inertness offluorocarbons, minimum storage restrictions are needed for such gels,making them suited for anti-STD applications in developing countries. Anadditional advantage is that the ¹⁹F signal makes possible themeasurement of the in vivo distribution of such gels in the vagina using¹⁹F MRI. Currently, this is investigated by incorporating Gd³⁺-basedcontrast agents into the gel (E. S. Pretorius, K. Timbers, D. Malamud &K. Barnhart, Magnetic resonance imaging to determine the distribution ofa vaginal gel: before and after both simulated and real intercourse.Contraception, 66, 443-451, 2002; K. T. Barnhart, E. S. Pretorius, K.Timbers, D. Shera, M. Shabbout & D. Malamud, In vivo distribution of avaginal gel: MRI evaluation of the effects of gel volume, time andsimulated intercourse. Contraception, 70, 498-505, 2004). However,whether the image reflects the distribution of the gel or the contrastagents can be questionable (K. Barnhart, E. S. Pretorius, A. Stolpen &D. Malarmud, Distribution of topical medication in the human vaginal asimaged by magnetic resonance imaging. Fertility and Sterility, 76,189-195, 2001). One benefit of a fluorocarbon gel is that no extraneousMRI contrast agent is needed because the ¹⁹F signal of the gel itselfcan be used for MRI detection. In such a case, the image always belongsto the gel itself.

The present compounds, specifically the disclosed F-oils andF-surfactants, can be used in connection with the nanoemulsions. Thatis, F-oils and F-surfactants can be formulated into nanoemulsions. Sizeof fluorocarbon particles the nanoemulsions can be determined bysmall-angle X-ray scattering (SAXS). Microbicidal activities of theformulated nanoemulsions are assayed in cell culture first. To enhancesurface activity, the fluorocarbon moiety of each molecule is asexpanded as possible. Also, the shape of F-surfactants matches that ofF-oils so that any temporary “cavity” vacated by an F-oil can be filledin snugly by an F-surfactant and vice versa. This significantlyincreases the stability of the nanoemulsion. Structures of examplesuitable F-oils and F-surfactants are given in the scheme below.

The nanoemulsions can be formed simply by mixing the F-oil andF-surfactant with physiological buffer system and shaking vigorously.FIG. 6 shows pictures of one such formulation process (the amount ofF-surfactant increases from left to right). As can be seen, the emulsioneventually gels (right-most picture) as more surfactants are added. Theaverage particle size is 6.6 nm. Nanoemulsions of various surfacecharges and sizes can be formulated in a modular fashion byincorporating different F-oils and F-surfactants. Stability of thenanoemulsions can be followed by SAXS as a function of time and storagetemperature (i.e., to monitor particle size as a function of time).Virucidal assays are conducted against a series of viruses; includingrespiratory viruses (Flu A & B, measles, Rhino and SARS), STD viruses(HSV-1, -2, HHV-8 and HIV) and biodefense-related viruses (VEE, puntatoro, West Nile). Without wishing to be bound by theory, it is believedthat the nanoemulsions also have bactericidal activities.

M. FLUOROUS MIXTURE SYNTHESIS OF FLUORINATED DENDRON AMPHILES WITH ONESINGLET ¹⁹F NMR SIGNAL

Fluorinated microemulsions have a long history in biomedicine as, forexample, blood substitutes, ultrasound contrast agents, ¹⁹F nuclearmagnetic resonance (MR) imaging reagents, and tumor relaxometry andradiosensitization probes. Fluorocarbon liquid nanoparticles, formulatedas microemulsions, can also be employed as multifunctional drug deliveryvehicles for ¹⁹F magnetic resonance image (MRI) guided targeted cancertherapy. [See, e.g., Yu, Y. B. J. Drug Targeting 2006, 14, 663-669;Lanza, G. M.; Wickline, S. A. Prog. Cardiovasc. Dis. 2001, 44, 13-31;Morawski, A. M.; Winter, P. M.; Yu, X.; Fuhrhop, R. W.; Scott, M. J.;Hockett, F., Robertson, J. D.; Gaffaey, P. J.; Lanza, G. M.; Wickline,S. A. Magn. Reson. Med. 2004, 52, 1255-1262; Krafft, M. P. Adv. Drug.Del. Rev. 2001, 47, 209-228; Riess, J. G. Tetrahedron, 2002, 58,4113-4131; Kuznetsova, I. N. Pharmaceutical Chem. J. 2003, 37, 415-420.]However, because fluorinated amphiles A are highly hydrophobic and onlyslightly soluble in water, fluorinated amphiles A typically do not formfluorinated oil-in-water microemulsions. A class of fluorinated amphiles(G3 for an example) was then prepared.

In order to achieve high ¹⁹F signal intensity (twenty-seven chemicallyidentical fluorine atoms), the perfluoro-tert-butyl ether moiety wasselected. To increase the water solubility and biocompatibility, anamide-based dendron structure with tetra(ethylene glycol) ending groupswas conjugated to the fluorinated part with an amide bond. For asystematic screen and properties study, a few continual generations offluorinated dendron amphiles were preferred.

Fluorous mixture synthesis (FMS)—in which members of a series ofsubstrates are tagged with different fluorous tags, mixed, carriedthrough a series of reactions, and then separated based on the tag priorto detagging—provides a fast and convenient strategy for the synthesisof enantiomers, diastereomers and compound libraries. However, FMS hasnot before been used in connection with dendrimer synthesis. Here, EMSis employed in connection with the synthesis of the above mentionedfluorinated dendron amphiles. In this case, no detagging step istypically required, as the final products comprise fluorine. Thedemixing of different generations of fluorinated dendron amphiles can bebased on the size of nonfluorinated portion instead of differentfluorinated tags. As final products can be separated (or purified) byHPLC with a fluorinated column, each generation of fluorinated dendronscan be isolated in substantially pure form.

^(i)Conditions: (a) KH (35%), BrCH₂CO₂ ^(t-)Bu, THF, rt.; (b) TFA,anisol, CH₂Cl₂, rt.; (c) DIC, HOBt, HN(CH₂CO₂ ^(t-)Bu)₂, DMF/THF (1/1),rt.

All the intermediates for five generations of fluorinated dendrons werethen synthesized on a 300-mg scale in six steps from the common startingmaterial perfluoro-tert-butyl ether 5 in high yield without any columnpurification. Treatment of alcohol 5 with potassium hydride andtert-butyl bromoacetate gave ester 13 with 78% yield after simple phaseseparation of the quenched reaction mixture. Ester 13 was reacted withtrifluoroacetic acid to give the acid 14 in quantitative yield afterremoval of reaction solvent, anisol, and TFA. Acid 14 was then coupledwith di-tert-butyl iminodiacetate to yield ester 15 after fluorous solidphase extraction. By repeating the coupling and deprotecting processes,intermediates for the other three generations of dendrons were obtainedon 400-mg scales in excellent yields.

Fluorous mixture synthesis was then carried out by mixing the fouresters (13, 15, 17 and 19). The mixture of esters was then cleaved withTFA to give a mixture of acids, which was then coupled withdi-tert-butyl iminodiacetate to yield a mixture of higher generationesters after fluorous solid phase extraction. After addition of anotherpotion of ester 13, the new mixture of ester was then exposed todeprotection conditions, and the resulting acids were then coupled withtetra(ethylene glycol) derivative 20 to yield a mixture of benzyl ethers(M0-M4). Hydrogenolysis of the benzyl ether mixture gave a mixture offive fluorinated dendrons and their side products. The mixture can thenbe separated by HPLC on FluoroFlash® column to give each generation ofpure fluorinated dendron amphile on 300-mg scales, except generationfour. The overall yield after chromatographic purification is high forthe first four generations (G0 (87%), G1 (88%), G2 (81%), and G3 (71%)).For the G4, the target compound and sideproducts formed an inseparablemixture by HPLC.

With the four generations of fluorinated dendron amphiles in hand, someNMR experiments were carried out on G0-G3. All twenty-seven fluorineatoms in each generation of amphile give only one sharp singlet ¹⁹F NMRpeak, which is ideal for ¹⁹F MR imaging (FIG. 5 a). Also, T1 and T2 ofG0-G3 increase with molecular weight (FIG. 8 b).

N. FLUORINATED DENDRONS AS DRUG DELIVERY VEHICLES

In one aspect, fluorinated dendrons can be prepared according to Schemes3-9.

Starting material 13 (4.0 mmol, 3.62 g) was subjected to deprotectionand coupling to provide pure 16 (Flash chromatography on F-silica gelpurification gave 16 (3.96 g, 92% over all yield, 3.68 mmol)). Aftertransferral of a portion of compound 16 (0.40 mmol, 430 mg) to a flaskA, the remainder of compound 16 (3.27 mmol, 3.52 g) was then subjectedto deprotection and coupling to provide pure compound 17 (Flashchromatography F-silica gel purification gave 17 (4.22 g, 2.98 mmol, 91%overall yield)). After transferral of a portion of compound 17 (0.3mmol, 425 mg) to the flask A, the remainder of compound 17 (3.79 g, 2.68mmol) was then subjected to deprotection and coupling to provide purecompound 19 (Flash chromatography F-silica gel purification gave 19(4.84 g, 86% overall yield, 2.30 mmol)). After transferral of portionsof compound 19 (0.2 mmol, 421 mg) and compound 13 (0.6 mmol, 543 mg)into the flask A, the mixture of starting material was put into themixture synthesis.

Reaction Conditions Starting Material 13 13 16 17 19 MW MW MW MW MW904.35 904.35 1075.54 1417.93 2102.71 Input of SM 0.6 mmol 0.5 mmol 0.4mmol 0.3 mmol 0.2 mmol 542.61 mg 452.18 mg 430.22 mg 425.38 mg 420.54 mgStep 1 Amount of ester 0.5 mmol 0.8 mmol 1.2 mmol 1.6 mmol (4.1 mmoltotal) Deprotection DCM (24 mL); TFA (16 mL), Anisol (1 mL), rt 2 h Workup Removal of solvent, the residue was evaporated with toluene (30 mL)twice to dryness and put into the next step. Step 2 Amount of acid (4.1mmol 0.5 mmol 0.8 mmol 1.2 mmol 1.6 mmol total) HN(CH₂CO₂ ^(t-)Bu)₂ 1.5mmol 2.4 mmol 3.6 mmol 4.8 mmol MW 245.32; 3 eq. 12.3 mmol, 3.02 g DICMW 126.2, 1.5 mmol 2.4 mmol 3.6 mmol 4.8 mmol d = 0.815; 3 eq. 12.3mmol, 1.552 g, 1.9 mL HOBt MW 135.1, 3 1.5 mmol 2.4 mmol 3.6 mmol 4.8mmol eq. 12.3 mmol, 1.662 g Reaction in DMF (40 mL) overnight. Workup 1. Addition of 6 mL water to the reaction mixture 2. Load to theF-silica gel column (100 g) 3. Washed with MeOH/Water (8:2, 100 mL) 4.Washed with MeOH/TFE (8/2, 200 mL) and colleted the fraction 5. Washedwith Acetone (200 mL 6. collected 4 & 5, removal of solvent to drynessStep 3 Amount of ester 0.6 mmol 1.0 mmol 1.6 mmol 2.4 mmol 3.2 mmol (8.8mmol total) Deprotection DCM (48 mL); TFA (32 mL), rt 2 h (Twice) Workup Removal of solvent, the residue was evaporated with toluene (30 mL)to dryness and put into the next step. Step 4 Amount of acid (8.8 mmol0.6 mmol 1.0 mmol 1.6 mmol 2.4 mmol 3.2 mmol total) H₂N(CH₂CH₂O)₄Bn 1.8mmol 3.0 mmol 4.8 mmol 7.2 mmol 9.6 mmol MW 283.4, 26.4 mmol, 7.482 gDIC MW 126.2, 1.8 mmol 3.0 mmol 4.8 mmol 7.2 mmol 9.6 mmol d = 0.815; 3eq. 26.4 mmol, 3.332 g, 4.01 mL HOBt MW 135.1, 3 1.8 mmol 3.0 mmol 4.8mmol 7.2 mmol 9.6 mmol eq. 26.4 mmol, 3.567 g Reaction in DMF (50 mL)overnight. Work up 1. Addition of 6 mL water to the reaction mixture 2.Load to the F-silica gel column (100) 3. Washed with MeOH/Water (8:2, 30mL) 4. Washed with MeOH (40 mL) and colleted the fraction 5. Washed withAcetone (40 mL) Step 5 To a stirred solution of crude products inMethanol 100 mL was added Pd/C (2 g). After degassed for 5 min, theresulting suspension was stirred under an atmosphere of hydrogen gasovernight. After filtrated though a pad of Celite, the solution wasconcentrated under vacuum and purified by HPLC to give the 5 products.

O. HIGHLY FLUORINATED CHELATORS FOR ¹H-¹⁹F MULTINUCLEAR MAGNETICRESONANCE IMAGING

Multinuclear MR imaging, such as ¹H-³¹P and ¹H-²³Na, is playing anincreasingly important role in cancer research and holds great potentialfor cancer diagnosis and intervention. Compared with other nuclei, ¹⁹Fhas a sensitivity of only second to ¹H (83% as sensitive) and hasnegligible background interference in human tissues. Thus, areas ofinterest can be quickly identified based on Gd(M)-enhanced ¹H₂O signals,and a more accurate quantification can be based on the ¹⁹F signal, byusing a ¹H-¹⁹F multinuclear MR reagent with the same imaging modality.

When conjugated to a drug, local drug concentration can be measured by¹⁹F MRI and hence an image-based dosing for each patient (individualizeddoimetry) can be developed. Moreover, ¹⁹F MR relaxometry is awell-established non-invasive method for oxygen tension determinationand is the only NMR technique to determine the absolute value of oxygentension. The significance of oximetry in radiotherapy lies in the factthat oxygen tension plays a role in tumor hypoxia and sensitivity towardradiation.

However, conventional ¹⁹F MR imaging studies typically employ eitherlinear fluorocarbons (such as perfluorooctyl bromide), which exhibit amultipeak-spectra resulting in ¹⁹F MR imaging with chemical shiftartifacts, or perfluoro-15-crown-5, which —CF₂— groups are significantlyless sensitive than —CF₃ group toward oxygen tension in ¹⁹F oximetry.Therefore, the synthesis of novel —CF₃ group rich fluorinated MR imagingreagents with a single sharp resonance peak is desirable to optimizecurrent ¹⁹F MR imaging study.

Two highly fluorinated DOTA derivatives (35 and 1) were prepared. Thesestructures were selected based upon the following criteria: Firstly,cyclic chelator DO3A was chosen for ¹H MRI since it has the higheststability toward Gd(M) among the chelators currently used in clinic.Secondly, perfluoro-tert-butyl ether was used for ¹⁹F MR imaging becauseof its high ¹⁹F signal intensity (twenty-seven chemical identicalfluorine atoms) and high stability (stable to base and acid). Finally, atetraethylene glycol chain was introduced in compound 1 to avoid thepossibility of the bulky and highly hydrophobic fluorinated ether parthampering the chelation process and reduce the water solubility.

The synthesis of target molecules 35 and 1 commenced with thepreparation of common building block 5 (Scheme 10). Protection ofpentaerythritol 2 as the orthoacetate, followed by protection of thefourth hydroxyl group with benzyl bromide and hydrolysis of theorthoacetate, finished the corresponding triol 3. After triol 3 wasreacted with nonafluoro-tert-butanol to provide the perfluoro-tert-butylether 4, the benzyl group in ether 4 was removed by aluminum chloride toafforded desired heavy fluorinated alcohol 5 on a 50-gram scale.

With building block 5 in hand, the target molecule 1 was thensynthesized (Scheme 11). Treatment of alcohol 5 withtrifluoromethanesulfonic anhydride in the presence of pyridine gavetrifluoromethanesulfonate 36 with excellent yield by simply phaseseparation after the addition of a small amount of water to the reactionmixture.

Tetrahydrofuran is an excellent solvent for this reaction due to goodsolubility of alcohol 5. The fluorinated fragment was then conjugated tothe cyclene ring by reaction of trifluoromethanesulfonate 36 with twoequivalents of cyclene in a mixture of tetrahydrofuran anddichloromethane (1/1) at 60° C. overnight. The pure product 37 wasisolated by simple F362 extraction with an 88% yield. The reaction ofcompound 37 with ethyl bromoacetate in the presence of potassiumcarbonate in a mixture of tetrahydrofuran/dimethylformamide (1/1) at 60°C. provided triethylester 38 with an excellent yield. Finally,hydrolysis of the triethylester with lithium hydroxide in water andmethanol afforded 35 with a 97% yield.

Linker 8′ was synthesized in good yield on a 50-gram scale fromcommercially available tetraethylene glycol 6 by selective protection ofone of the hydroxyl groups with benzyl bromide and transformation of theother hydroxyl group into the corresponding methanesulfonate (Scheme12).

In the synthesis of DOTA 1 (Scheme 13), the heavily fluorinated alcohol5 was first attached to the hydrophilic tetraethylene glycol chain toprovide compound 9 in good yield by treating the alcohol 5 withpotassium hydride in tetrahydrofuran at room temperature for 30 minutes,then slowly addition of the methanesulfonylate 8′ at the sametemperature. Due to the three bulky perfluoro-tert-butyl group incompound 5, use of sodium hydride as a base resulted in recovery of thealcohol 5 and methanesulfonylate 8′ only after a long reaction time.Then, removal of the benzyl group in compound 9 by palladium hydroxidecatalyzed hydrogenolysis gave alcohol 10 with an excellent yield, whichwas then treated with methanesulfonyl chloride and triethyl amine togive the methanesulfonylate 11 in a quantitative yield. Attaching thecyclene ring to the fluorinated moiety was achieved by treating compound11 with two equivalents of cyclene at 60° C. Purification of theresulting cyclene derivative 12 from the reaction mixture was laborious.Then solid phase extraction on fluorinated silica gel was employed, andcompound 12 was isolated with a 93% yield. Compound 12 was reacted withethyl bromoacetate in the presence of potassium carbonate indimethylformamide at 60° C. provided tri-ethyl ester 13 with anexcellent yield. Finally, treatment of compound 13 with lithiumhydroxide in water and methanol gave 1 with a 99% yield.

P. HIGHLY FLUORINATED DENDRON CHELATORS

Using methods analogous to those disclosed herein, highly fluorinateddendron chelators, which combine features of fluorinated dendrons asdrug delivery vehicles and highly fluorinated chelators for ¹H-¹⁹Fmultinuclear magnetic resonance imaging, can be prepared. Exemplarycompounds include:

Q. EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Unless otherwise indicated, reference numbers used in the syntheticprocedures refer to the immediate schematic diagram.

1. Synthesis of Surfactant with —OH Ending Group

a. Synthesis of Compound 3

To a suspension of pentaerythritol 2 (21.2 g, 156 mmol) in toluene (20mL) was added triethyl orthoacetate (25.3 g, 156 mol) andp-toluenesulfonic acid monohydrate (100 mg). The resulting mixture wasgradually heated with an oil bath, and ethanol was slowly distilled fromthe mixture overnight. After all ethanol had distilled, the bathtemperature was raised to 125° C. and toluene was distilled off untilthe solution was homogeneous. The solution was allowed to cool and theresidue was used in the next step without further purification. PowderedKOH (41.2 g, 734.3 mmol) was suspended in dimethyl sulfoxide (250 mL),and this mixture was stirred at room temperature for 15 min. The residuefrom the former step (25.0 g, 156 mmol) was added in one portionfollowed quickly by benzyl bromide (32.2 g, 188.0 mmol). The reactionmixture (which became quite hot) was stirred for 4 hours and thendiluted with water (2500 mL) and extracted with diethyl ether (250 mL).The combined extracts were washed with brine (50 mL) and water (50 mL),dried over MgSO₄, concentrated to dryness and flash chromatography toafford 39.0 g of intermediate. The intermediate was dissolved inmethanol (100 mL) and treated with 0.01 N HCl (400 mL). The resultingmixture was stirred at 25° C. for 1 h, treated with sodium bicarbonate(14.5 g, 173.0 mmol), stirred for an additional 1 h, and concentrated.Trituration of the resulting solid residue with methanol (200 mL) andconcentration of the triturate afforded 19.4 g (55%) of compound 3 as acolorless viscous oil. ¹H NMR (CD₃OD) δ 3.34 (s, 2H), 3.55 (s, 6H), 4.37(s, 2H), 7.17-7.33 (m, 5H).

b. Synthesis of Compound 4

To a mixture of compound 3 (30.1 g, 128 mmol), triphenylphosphine (150.7g, 575 mmol) and 4 Å molecular sieve (30.0 g) in tetrahydrofuran (500mL) at 0° C. was added dropwise diethylazodicarboxylate (100.0 g, 575mmol). After the addition, the reaction mixture was allowed to warm toroom temperature and stirred for additional 20 minutes. Thenperfluoro-tert-butanol (135.7 g, 575 mmol) was added in one portion andthe resulting mixture was stirred at 45° C. for 30 hours. The mixturewas evaporated to dryness and dissolved in dichloromethane (600 mL). Theresulting mixture was extracted with perfluorohexane (200 mL, 3 times).The combined extraction was washed with dichloromethane (50 mL) andconcentrated to give compound 4 as a clear oil (90.5 g, 81%). ¹H NMR(400 MHz, CDCl₃) δ 3.45 (s, 2H), 4.08 (s, 6H), 4.47 (s, 2H), 7.25-7.35(m, 5H); ¹⁹F NMR (376 MHz, CDCl₃) δ−73.49 (s); ¹³C NMR (100.7 Hz, CDCl₃)δ 46.43, 65.49, 65.62, 73.94, 79.70 (q, J=29.9 Hz), 120.33 (q, J=292.5Hz), 127.99, 128.11, 128.57, 137.34; MS (CI) m/z 881 (M⁺+1, 31), 880(M⁺, 11), 803 (26), 91 (100); HRMS (CI) Calcd for C₂₄H₁₅F₂₇O₄: 880.0539,Found: 880.0515.

c. Synthesis of Compound 5

To a stirred mixture of compound 4 (90.4 g, 102.7 mmol) and anisole(44.4 g, 410.9 mmol) in dichloromethane (500 mL) at 0° C. was slowlyadded powdered aluminum chloride anhydrous (41.1 g, 308.1 mmol). Afterthe addition, the reaction mixture was allowed to warm to roomtemperature and stirred for additional 30 minutes. The reaction mixturewas quenched by slowly addition of 1N HCl (100 mL) and the resultedmixture was extracted with perfluorohexane (200 mL, 3 times). Thecombined extraction was washed with dichloromethane (50 mL) andconcentrated to give compound 5 as a clear oil (80.3 g, 99%). ¹H NMR(400 MHz, Acetone-d6) δ 3.73 (d, J=4.0 Hz, 2H), 4.27 (s, 6H), 4.44 (t,J=4.0 Hz, 1H); ¹⁹F NMR (376 MHz, Acetone-d6) δ−71.20 (s); ¹³C NMR (100.7MHz, Acetone-d6) δ 52.05, 62.84, 71.65, 84.75 (q, J=29.9 Hz), 122.77 (q,J=291.8 Hz); MS (CI) m/z 791 (M⁺+1, 8), 91 (100); HRMS (CI) Calcd forC₁₇H₁₀F₂₇O₄: 791.0149, Found: 791.0131.

d. Synthesis of Compound 7

To a stirred mixture of tetraethylene glycol 6 (66.0 g, 340 mmol),imidazole (33.9 g, 498 mmol) and 4-dimethylaminopyridine (5 g, 41 mmol)in dichloromethane (1000 mL) and dimethylformamide (100 mL) at 0° C. wasslowly added a solution of tert-Butylchlorodimethylsilane (25.1 g, 166mmol) in dichloromethane (100 mL) over 4 hours. The resulted mixture wasstirred overnight. The reaction mixture was quenched with 1N HCl (300mL), extracted with ethyl acetate. The combined organic phase was driedover magnesium sulfate, concentrated to dryness, and purified by flashchromatography to afford compound 7 (44.5 g, 87%) as clear oil. ¹H NMR(400 MHz, CDCl₃) δ 0.00 (s, 6H), 0.83 (s, 9H), 1.79 (s, 1H), 3.45-3.70(m, 16H).

e. Synthesis of Compound 8

To a stirred mixture of 7 (44.5 g, 145 mmol) and triethylamine (58.4 g,578 mmol) in dichloromethane (700 mL) at 0° C. was added methanesulfonylchloride (33.1 g, 289 mmol). After the addition, the reaction mixturewas stirred overnight at room temperature. The reaction mixture wasquenched with 1N HCl (300 mL), extracted with ethyl acetate. Thecombined organic phase was dried over magnesium sulfate, concentrated todryness and flash chromatography to afford compound 8 (55.3 g, 99%) asclear oil. ¹H NMR (400 MHz, CDCl₃) δ 0.00 (s, 6H), 0.83 (s, 9H), 3.01(s, 3H), 3.48 (t, J=5.2 Hz, 2H), 3.55-3.61 (m, 8H), 3.68-3.71 (m, 4H),4.29-4.32 (m, 2H).

f. Synthesis of Compound 39

To a stirred mixture of 5 (23 g, 29 mmol) in tetrahydrofuran (150 mL) at0° C. was treated with potassium hydride (7.0 g, 25% on mineral oil, 44mmol) and the resulting mixture was stirred for additional 1 hour atroom temperature. A solution of compound 7 (17.0 g, 44 mmol) intetrahydrofuran (20 mL) was then added and the resulting mixture wasstirred overnight. The reaction mixture was quenched with 1N HCl (100mL), extracted with ethyl acetate. The combined organic phase was driedover magnesium sulfate, concentrated to dryness and flash chromatographyto afford compound 39 (17.2 g, 61%) as clear oil. ¹H NMR (400 MHz,CDCl₃) δ 0.00 (s, 6H), 0.833 (s, 9H), 3.41 (s, 2H), 3.49-3.60 (m, 14H),3.71 (t, J=5.6 Hz, 2H), 4.01 (s, 6H); ¹⁹F NMR (376 MHz, CDCl₃) δ−73.19(s); ¹³C NMR (100.7 Hz, CDCl₃) δ−5.58, 0.37, 18.25, 25.71, 46.23, 62.72,65.52, 66.37, 70.30, 70.62, 70.70, 70.75, 70.79, 72.69, 79.52 (q, J=30.0Hz), 120.17 (q, J=292.5 Hz);

g. Synthesis of Compound 10

To a stirred mixture of 39 (17.2 g, 15.9 mmol) in tetrahydrofuran (100mL) at 0° C. was treated with tetrabutylammonium fluoride (20 mL, 1Nsolution in THF, 20 mmol) and resulting mixture was stirred foradditional 2 hour at room temperature. The reaction mixture wasconcentrated to dryness and flash chromatography to afford compound 10(12.8 g, 83%) as clear oil. ¹H NMR (400 MHz, CDCl₃) δ 3.39 (s, 2H),3.53-3.60 (m, 16H), 3.99 (s, 6H); ¹⁹F NMR (376 MHz, CDCl₃) δ−73.49 (s);¹³C NMR (100.7 Hz, CDCl₃) δ 46.22, 58.78, 61.69, 65.55, 66.41, 70.30,70.32, 70.54, 70.65, 70.76, 72.49, 79.51 (q, J=36.7 Hz), 120.16 (q,J=292.6 Hz), MS (CD) m/z 967 (M⁺+1, 100); HRMS (CI) Calcd forC₂₅H₂₆F₂₇O₈: 967.1150, Found: 967.1173.

When n is 1, 2, 3, 5, 6, 7, 8, the compounds can also be synthesized bythe same procedure as compound 10. When n is larger than 8, the PEG isin a form of a mixture. Fluorinated Surfactant with such PEG group canalso be synthesized with the same procedure as outlined for compound 10.

Such compound can also be synthesized with the same procedure asoutlined for compound 10.

2. Synthesis of Surfactant with —NR₂ Ending Group

a. Synthesis of Compound 11

To a stirred mixture of 10 (15.5 g, 16 mmol) and triethylamine (6.5 g,64 mmol) in dichloromethane (100 mL) at 0° C. was added methanesulfonylchloride (3.64 g, 32 mmol). After the addition, the reaction mixture wasstirred overnight at room temperature. The reaction mixture was quenchedwith 1N HCl (100 mL), extracted with ethyl acetate. The combined organicphase was dried over magnesium sulfate, concentrated to dryness andflash chromatography to afford compound 11 (15.8 g, 95%) as clear oil.¹H NMR (400 MHz, CDCl₃) δ 3.04 (s, 3H), 3.43 (s, 2H), 3.53-3.58 (m, 8H),3.61-3.67 (m, 4H), 3.73-3.76 (m, 2H), 4.04 (s, 6H), 4.35-4.37 (m, 2H);¹⁹F NMR (376 MHz, CDCl₃) δ−73.37 (s); ¹³C NMR (100.7 Hz, CDCl₃) δ 37.54,46.18, 63.53, 65.45, 66.31, 68.99, 69.09, 69.22, 70.23, 70.52, 70.58,70.72, 79.46 (q, J=30.7 Hz), 120.11 (q, f=294.1 Hz); MS (Maldi) m/z 1067(M+Na⁺, 100); HRMS (Maldi) Calcd for C₂₆H₂₇F₂₇O₁₀SNa: 1067.0792, Found:1067.0755.

b. Synthesis of Compound 40

A mixture of compound 11 (15.8 g, 15.2 mmol) and sodium azide (2.0 g,30.4 mmol) in dimethylformamide (100 mL) was stirred at 60° C. for 6hour. Removal of solvent, the residue was purified by flash columnchromatography on silica gel to give compound 40 as a liquid (13.1 g,87%).

c. Synthesis of Compound 41

A mixture of palladium on carbon (400 mg) in methanol (20 μL) wasdegassed for 1 minute and stirred under an atmosphere of hydrogen for 30minute. The compound 40 (1.92 g, 1.94 mmol) in methanol (5 mL) was addedto the mixture and the resulting mixture was stirred under hydrogenatmosphere overnight. After filtration, the mixture was concentratedunder vacuum and purified by flash column chromatography on silica gelto compound 41 as a liquid (1.17 g, 63%).

3. Synthesis of Surfactant with a —SH Ending Group

a. Synthesis of Compound 42

Potassium thioacetate (1.71 g, 15 mmol) was added to a stirred mixtureof compound 11 (6.76 g, 7 mmol) in dimethylformamide (50 mL). Theresulting mixture was stirred at 50° C. overnight. After removal ofsolvent under vacuum, the residue was purified by flash columnchromatography on silica gel to give compound 42 as a liquid (5.91 g,83%).

b. Synthesis of Compound 43

To a stirred mixture of compound 42 (4.7 g, 4.6 mmol) in methanol (20mL) was added 1N sodium hydroxide (15 mL) and the resulting mixture wasstirred at room temperature for 5 hours. Then 2N hydrochloric acid (8mL) was added. After removal of solvent, the residue was purified byflash column chromatography on silica gel to give compound 43 as aliquid (4.18 g, 93%).

Such compound can also been synthesized with the same procedure asoutlined for compound 43.

4. Synthesis of Surfactant with a —COOH Ending Group

To a stirred mixture of compound 10 (3.87 g, 4 mmol) in acetone at 0° C.was added dropwise a solution of Jones Reagent (3N, 12 mL). After theaddition, the mixture was stirred at room temperature for additional 2hours. Removal solvent under reduced pressure, the residue was purifiedby flash column chromatography on silica gel to give compound 45 as aviscous oil (3.30 g, 84%).

Such compound can also be synthesized with the same procedure asoutlined for compound 45.

5. Synthesis of Surfactant with a Chelator Ending Group

a. Synthesis of Compound 12

To a stirred solution of compound 11 (10.3 g, 9.8 mmol) indimethylformamide (100 mL) was added cyclen (3.48 g, 20 mmol) in oneportion. Then the mixture was stirred at 60° C. overnight. After removalof solvent under vacuum, the residue was purified by flash columnchromatography on basic aluminum oxide to compound 12 as viscous oil(10.2 g, 93%).

b. Synthesis of Compound 50

To a stirred solution of compound 12 (9.8 g, 8.8 mmol) intetrahydrofuran (50 mL) and dimethylformamide (80 mL) was addedanhydrous potassium carbonate (9.66 g, 70 mmol) and ethyl bromoacete(7.3 g, 43.8 mmol). The resulting mixture was stirred overnight at 60°C. Then the mixture was washed with brine (200 mL), the aqueous layerwas extracted with ethyl acetate (100 mL, 4 times). The combined layerswere dried over magnesium sulfate, concentrated under vacuum. Theresidue was purified by flash column chromatography on silica gel togive the compound 50 as viscous oil (9.95 g, 82%).

c. Synthesis of Compound 1

Lithium hydroxide (622 mg, 28 mmol) was added to a solution of compound13 (5.52 g, 4 mmol) in tetrahydrofuran (100 mL), methanol (100 mL) andwater (100 mL). The resulting mixture was stirred at room temperaturefor 8 hours. Then 1N hydrochloride acid was added to adjust the solutionto pH 3. Removal of solvent under vacuum, the residue was purified byflash column chromatography on aluminum oxide to give compound 1 as asolid (5.0 g, 97%).

When n is other than 4, such as 0, 1, 2, 3, 8, such surfactants can alsobe synthesized in the same way as for 1.

6. Synthesis of Surfactant with a Peptide as an Ending Group:

The fluorinated surfactant 45 was incorporated into peptide during thesolid phase peptide synthesis as a terminal amino acid by employ therouting coupling, cleavage and deprotection procedure.

Such compounds can also be synthesized as outlined above.

7. Synthesis of Surfactant with a Nitrogen Atom as Branch Point in aHydrophobic Moiety

a. Synthesis of Compound 7′

An 60% dispersion of Sodium hydride (20.8 g, 0.52 mol) in paraffin waswashed twice with tert-butyl methyl ether and decanted; the sodiumhydride was suspended in tetrahydrofuran (300 mL), and then a mixture oftetraethylene glycol 6 (97 g, 0.5 mol) and tetrahydrofuran (50 mL) wasadded dropwise. After the evolution of hydrogen had stopped, benzylbromide (51.2 g, 0.3 mol) was added and the reaction mixture stirred for2 h. Water was added, the organic layer separated, and the aqueous phaseextracted with tert-butyl methyl ether. The combined organic phases weredried and the solvents removed under reduced pressure. The crude product7′ (76.5 g, 90%) was used in the next step without further purification.

b. Synthesis of Compound 8′

To a stirred mixture of 7′ (45.1 g, 159 mmol) and triethylamine (62.2 g,636 mmol) in dichloromethane (800 mL) at 0° C. was added methanesulfonylchloride (36.4 g, 318 mmol). After the addition, the reaction mixturewas stirred overnight at room temperature. The reaction mixture wasquenched with 1N HCl (300 mL), extracted with ethyl acetate. Thecombined organic phase was dried over magnesium sulfate, concentrated todryness and flash chromatography to afford compound 8′ (56.2 g, 98%) asclear oil.

c. Synthesis of Compound 47

To a mixture of compound 8′ (36.2 g, 100 mmol) in chloroform (800 mL)was added commercial available 46 (52.5 g, 500 mmol) and the resultingmixture was stirred at 40° C. overnight. Then removal solvent undervacuum, the residue was purified by flash column chromatography onsilica gel to give compound 48 as a liquid (19.7 g, 53%).

d. Synthesis of Compound 48

To a mixture of compound 47 (18.6 g, 50 mmol), triphenylphosphine (39.5g, 150 mmol) and 4 Å molecular sieve (15.0 g) in tetrahydrofuran (200mL) at 0° C. was added dropwise diethylazodicarboxylate (26.1 g, 150mmol). After the addition, the reaction mixture was allowed to warm toroom temperature and stirred for additional 20 minutes. Thenperfluoro-tert-butanol (35.4 g, 150 mmol) was added in one portion andthe resulting mixture was stirred at 45° C. for 30 hours. The mixturewas evaporated to dryness and purified by flash column chromatography onsilica gel to give compound 48 as a clear oil (24.6 g, 61%).

e. Synthesis of Compound 49

A mixture of compound 48 (12.1 g, 15 mmol) and palladium on carbon (1.2g) in methanol (80 mL) was degassed for 1 minute. Then the mixture wasstirred under an atmosphere of hydrogen for 12 hours. After filtration,the filtrate was concentrated under vacuum and the residue was purifiedby flash column chromatography on silica gel to give compound 49 asclear oil (10.2 g, 95%).

-   -   ((CF₃)₃COCH₂CH₂)₂N(CH₂CH₂O)nH((CF₃)₃C CH₂CH₂)₂N(CH₂CH₂O)nCH₂X

8. Synthesis of Fluorinated Dendrons

a. Alcohol 7′

To a stirred solution of tetraethylene glycol 6 (97.0 g, 500.0 mmol) intetrahydrofuran (450 mL) at 0° C. was added sodium hydride (60% inparaffin, 20.8 g, 520.0 mmol) slowly and the resulted mixture wasstirred at rt. for 30 min. Then benzyl bromide (51.2 g, 300.0 mmol) wasadded and the resulted mixture was stirred at rt. overnight. Afterquenched the reaction with water (200 mL), the mixture was extractedwith ethyl acetate (100 mL, 4 times). The combined organic phase wasdried over anhydrous magnesium sulfate. After concentration undervacuum, the residue was purified by flash chromatography on silica gel(n-hexane/ethyl acetate=1/1) to give alcohol 7′ as clear oil (76.5 g,90%). ¹H NMR (400 MHz, CDCl₃) δ 1.28-7.29 (m, 5H), 4.51 (s, 2H),3.52-3.66 (m, 16H).

b. Methanesulfonate 8′

To a stirred solution of alcohol 7′ (69.3 g, 243.7 mmol) andtriethylamine (49.2 g, 68.4 mL, 487.0 mmol) in CH₂Cl₂ (700 mL) at 0° C.was added methanesulfonyl chloride (41.9 g, 28.3 mL, 365.6 mmol). Theresulted mixture was stirred at rt. overnight and quenched with water(400 mL). Organic phase was collected and the aqueous phase wasextracted with ethyl acetate. The combined organic phase was washed with2N HCl (100 mL), brine (100 mL) and dried over anhydrous magnesiumsulfate. Concentrated the solution under vacuum gave themethanesulfonate 8′ as clear oil (86.6 g, 98%). ¹H NMR (400 MHz, CD₃Cl₃)δ 7.25-7.32 (m, 5H), 4.54 (s, 2H), 4.32-4.34 (m, 2H), 3.71-3.73 (m, 2H),3.59-3.66 (m, 12H), 3.03 (s, 3H).

c. Azide 23

The suspension of methanesulfonate 8′ (69.3 g, 191.2 mmol) and sodiumazide (16.2 g, 254.6 mmol) in dimethylformamide (800 mL) was stirred at110° C. overnight. After removal of the solvent under vacuum, theresidue was purified by plash chromatography on silica gel((n-hexane/ethyl acetate=2/1)) to give the azide 23 as clear oil (56.8g, 96% yield). ¹H NMR (400 MHz, CD₃OD) δ 7.20-7.27 (m, 5H), 4.49 (s,2H), 3.55-3.61 (m, 14H), 3.29 (t, J=5.0 Hz, 2H).

d. Amine 24

To a stirred solution of azide 23 (55.2 g, 178.4 mmol) in drytetrahydrofuran (700 mL) at 0° C. was added triphenyl phosphine (59.7,227.7 mmol) and the resulted mixture was stirred at rt. for 10 h. Water(5.8 mL, 323.4 mmol) then added to hydrolysis the intermediatephosphorous adduct. After 10 h, the reaction mixture was diluted withwater (1000 mL) and washed with toluene (200 mL, twice). Evaporation ofthe aqueous phase yielded the product (50.6 g, 91% yield). ¹H NMR (400MHz, CD₃OD) δ 7.21-7.29 (m, 5H), 4.51 (s, 2H), 3.55-3.64 (m, 12H), 3.43(t, J=5.2 Hz, 2H), 2.78 (t, J=5.2 Hz, 2H).

e. Tert-Butyl Ester 13

A suspension of potassium hydride (30%, 3.2 g, 24.0 mmol) was addedslowly to a stirred solution of alcohol 5 (15.8 g, 20.0 mmol) intetrahydrofuran (200 mL) at 0° C. After 10 min, tert-butyl bromoacetate(5.9 mL, 7.8 g, 40.0 mmol) was added to the suspension in one portion atrt and the resulted mixture was stirred at rt overnight. After quenchedthe reaction with water (20 mL), the mixture was transferred intoseparatory funnel and the lower phase was collected as clear oil.Removal of low boiling point impurities under vacuum gave the ester 13as clear oil (14.1 g, 78% yield). ¹H NMR (400 MHz, CD₃Cl₃) δ 4.14 (s,6H), 3.91 (s, 2H), 3.57 (s, 2H), 1.46 (s, 9H); ¹⁹F NMR (376 MHz, CD₃Cl₃)δ−73.51 (s); ¹³C NMR (100.7 MHz, CD₃Cl₃) δ 168.5, 120.2 (q, J=293.4 Hz),81.8, 79.3-80.0 (m), 69.2, 67.2, 66.2, 46.1, 27.9; MS (MALDI-TOF) m/z905 (M⁺+1, 100); HRMS (MALDI-TOF) calcd for C₂₃H₂₀F₂₇O₆ 905.0829, found905.0823.

f. Acid 14

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,Acetone-d6) δ 4.29 (s, 6H), 4.14 (s, 2H), 3.73 (s, 2H); ¹⁹F NMR (376MHz, Acetone-d6) δ−71.24 (s); ¹³C NMR (100.7 MHz, Acetone-d6) δ 170.9,121.2 (q, J=292.5 Hz), 80.1-81.0 (m), 68.6, 67.5, 67.1, 47.1; MS(MALDI-TOF) m/z 849 (M⁺+1, 100); HRMS (MALDI-TOF) calcd for C₁₉H₁₂F₂₇O₆849.0203, found 849.0197.

g. Amide 25

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃OD) δ 7.20-7.33 (m, 5H), 4.53 (s, 2H), 4.19 (s, 6H), 3.92 (s, 2H),3.54-3.66 (m, 12H), 3.52-3.54 (m, 4H), 3.42 (t, J=5.6 Hz, 2H); ¹⁹F NMR(376 MHz, CD₃OD) δ−71.13 (s); ¹³C NMR (100.7 m/z, CD₃OD) δ 168.1, 138.2,128.2, 127.6, 127.5, 120.0 (q, J=293.3 Hz), 79.2-79.9 (m), 73.1, 71.1,70.54, 70.5, 70.4, 70.3, 70.2, 69.6, 69.3, 66.7, 65.0, 46.1, 38.6; MS(MALDI-TOF) m/z 1136 (M⁺+1, 100); HRMS (MALDI-TOF) calcd forC₃₄H₃₄F₂₇NNaO₉ 1136.1700, found 1136.1669.

h. Surfactant 26

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃Cl₃) δ 4.03 (s, 6H), 3.89 (s, 2H), 3.54-3.64 (m, 12H), 3.43-3.54 (m,6H); ¹⁹F NMR (376 MHz, CD₃Cl₃) δ−73.44 (s); ¹³C NMR (100.7 MHz, CD₃Cl₃)δ 168.4, 120.0 (q, J=293.3 Hz), 79.4-79.9 (m), 72.4, 71.0, 70.4, 70.2,70.1, 70.08, 66.5, 64.9, 61.5, 46.2, 38.7; MS (MALDI-TOF) m/z 1046(M⁺+Na), 1024 (M⁺+1, 100); HRMS (MALDI-TOF) calcd for C₂₇H₂₈F₂₇NNaO₉1046.1231, found 1046.1220.

i. Di-Tert-Butyl Ester 15

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃Cl₃) δ 4.12 (s, 6H), 4.10 (s, 2H), 4.02 (s, 2H), 3.92 (s, 2H), 3.59(s, 2H), 1.44 (s, 9H), 1.42 (s, 9H); ¹⁹F NMR (376 MHz, CD₃Cl₃) δ−73.29(s); ¹³C NMR (100.7 MHz, CD₃Cl₃) δ 168.7, 167.9, 167.7, 120.1 (q,J=293.4 Hz), 82.8, 82.0, 69.9-79.8 (m), 69.5, 67.7, 66.5, 49.9, 48.6,46.0, 27.8, 27.76; MS (MALDI-TOF) m/z 1098 (M⁺+Na); HRMS (MALDI-TOF)calcd for C₃₁H₃₂F₂₇NNaO₉ 1098.1544, found 1098.1538.

j. Diacid 16

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃OD) δ 4.22 (s, 8H), 4.16 (s, 2H), 4.14 (s, 2H), 3.60 (s, 2H); ¹⁹F NMR(376 MHz, CD₃OD) δ−71.18 (s); ¹³C NMR (100.7 m/z, CD₃OD) δ 172.5, 172.1,171.7, 121.6 (q, J=293-4 Hz), 80.4-81.5 (m), 70.3, 68.6, 67.9, 50.0,47.4; MS (MALDI-TOF) m/z 986 (M⁺+Na), 964 (M⁺+1); HRMS (MALDI-TOF) calcdfor C₂₃H₁₇F₂₇NO₉ 964.0472, found 964.0476.

k. Diamide 27

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CDCl₃) δ 7.27-7.34 (m, 10H), 4.56 (s, 2H), 4.54 (s, 2H), 4.11 (s, 6H),4.03 (s, 2H), 3.86 (s, 2H), 3.84 (s, 2H), 3.60-3.69 (m, 24H), 3.54-3.58(m, 6H), 3.40-3.48 (m, 4H); ¹⁹F NMR (376 MHz, CDCl₃) δ-73.49 (s); ¹³CNMR (100.7 MHz, CDCl₃) δ 169.2, 169.1, 168.4, 138.1, 138.0, 128.33,128.3, 127.74, 127.7, 127.67, 127.6, 120.0 (q, J=292.5 Hz), 79.0-79.7(m), 77.2, 73.2, 73.16, 70.5, 70.46, 70.4, 70.27, 70.17, 70.08, 69.4,69.35, 69.3, 69.0, 68.9, 67.5, 66.1, 60.3, 53.1, 53.0, 46.0, 39.6, 39.2,20.9, 14.1; MS (MALDI-TOF) m/z 986 (M⁺+Na), 1516 (M⁺+1, 100); HRMS(MALDI-TOF) calcd for C₅₃H₆₂F₂₇N₃NaO₁₅ 1516.3647, found 1516.3599.

l. Surfactant 28

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃OD) δ 4.24 (s, 6H), 4.22 (s, 2H), 4.06 (s, 2H), 4.05 (s, 2H),3.58-3.67 (m, 22H), 3.54-3.57 (m, 8H), 3.39-3.43 (m, 4H); ¹⁹F NMR (376MHz, CD₃OD) δ−71.17 (s); ¹³C NMR (100.7 MHz, CD₃OD) δ 172.0, 171.4,170.8, 121.6 (q, J=292.5 Hz), 80.4-81.4 (m), 73.7, 71.6, 71.4, 71.3,71.2, 70.4, 70.3, 69.7, 68.7, 68.1, 62.2, 52.4, 47.3, 40.6, 40.4; MS(MALDI-TOF) m/z 1336 (M⁺+Na, 100); MS (MALDI-TOF) calcd forC₃₉H₅₀F₂₇N₃NaO₁₅ 1336.2708, found 1336.2703.

m. Tetra-Tert-Butyl Ester 17

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CDCl₃) δ 3.80-4.13 (m, 20H), 3.47 (s, 2H), 1.28-1.31 (m, 36H); ¹⁹F NMR(376 MHz, CDCl₃) δ−72.94 (s); ¹³C NMR (100.7 MHz, CDCl₃) δ 169.2, 169.1,168.2, 167.6, 167.5, 167.45, 167.3, 120.0 (q, J=293.4 Hz), 82.9, 82.6,81.8, 81.5, 78.7-79.8 (m), 68.3, 67.4, 66.6, 50.4, 50.1, 49.3, 48.9,47.6, 46.6, 45.8, 27.5, 27.4; MS (MALDI-TOF) m/z 1456 (M⁺+K); HRMS(MALDI-TOF) calcd for C₄₇H₅₈F₂₇KN₃O₁₅ 1456.3074, found 1456.3327.

n. Tetraacid 18

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃OD) δ 4.15 (s, 2H), 4.05-4.09 (m, 100H), 3.90-4.01 (m, 6H), 3.53-3.59(m, 2H), 3.43 (s, 2H); ¹⁹F NMR (376 MHz, CD₃OD) δ−71.13 (s); ¹³C NMR(100.7 MHz, CD₃OD) δ 172.5, 172.4, 172.3, 172.15, 172.0, 171.6, 171.0,121.6 (q, J=292.6 Hz), 80.4-81.5 (m), 69.7, 69.1, 68.7, 68.2, 67.5,50.5, 50.3, 49.8, 49.77, 47.3; MS (MALDI-TOF) m/z 1216 (M⁺+Na); HRMS(MALDI-TOF) calcd for C₃₁H₂₆F₂₇N₃NaO₁₅ 1216.0830, found 11216.0820.

o. Tetraamide 29

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CDCl₃) δ 7.25-7.34 (m, 20H), 4.55 (s, 8H), 4.07-4.15 (m, 14H), 3.90-3.95(m, 6H), 3.50-3.68 (m, 48H), 3.37-3.43 (m, 10H); ¹⁹F NMR (376 MHz,CDCl₃) δ−73.47 (s); ¹³C NMR (100.7 MHz, CDCl₃) δ 169.8, 169.7, 169.1,168.9, 168.7, 168.3, 168.0, 138.0, 128.3, 127.7, 127.6, 120.0 (q,J=293.3 Hz), 79.1-80.3 (m), 73.1, 70.4, 70.3, 70.0, 69.9, 69.8, 69.3,69.1, 69.0, 68.3, 67.4, 66.4, 52.8, 52.7, 52.5, 48.3, 47.2, 45.8, 39.3,39.2; MS (MALDI-TOF) m/z 2276 (M⁺+Na, 100); HRMS (MALDI-TOF) calcd forC₉₁H₁₁₈F₂₇N₇NaO₂₇ 2276.7542, found 2276.7494.

p. Surfactant 30

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃OD) δ 4.25 (s, 10H), 4.22 (s, 2H), 4.19 (s, 2H), 4.15 (s, 2H), 4.06(s, 2H), 4.05 (s, 2H), 3.60-3.60 (m, 42H), 3.54-3.58 (m, 16H), 3.44 (t,J=4.2 Hz, 4H), 3.38 (t, J=5.6 Hz, 4H); ¹⁹F NMR (376 MHz, CDCl₃) δ−73.48(s); ¹³C NMR (100.7 MHz, CDCl₃) δ 172.2, 171.9, 171.3, 171.2, 171.1,170.8, 170.5, 121.6 (q, J=292.5 Hz), 80.3-81.5 (m), 73.7, 71.6, 71.4,71.2, 71.18, 71.1, 70.4, 70.3, 69.5, 68.7, 68.3, 62.2, 53.2, 53.0, 52.5,49.7, 47.2, 40.5, 40.4; MS (MALDI-TOF) m/z 1916 (M⁺+Na, 100); HRMS(MALDI-TOF) calcd for C₆₃H₉₄F₂₇N₇NaO₂₇ 1916.5664, found 1916.5694.

q. Octa-Tert-Butyl Ester 19

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃OD) δ 4.40 (s, 2H), 4.34 (s, 2H), 4.28 (s, 4H), 4.25 (s, 8H),4.12-4.17 (m, 12H), 4.03-4.07 (m, 8H), 3.59 (s, 2H), 1.45-1.50 (m, 72H);¹⁹F NMR (376 MHz, CD₃OD) δ−71.16 (s); ¹³C NMR (100.7 MHz, CD₃OD) δ172.0, 171.6, 171.2, 171.1, 171.0, 170.8, 170.6, 169.7, 169.6, 169.55,169.43, 169.4, 121.6 (q, J=293.3 Hz), 84.1, 84.0, 83.8, 83.7, 83.1,83.0, 82.9, 80.3-81.5 (m), 69.5, 68.6, 68.3, 51.6, 51.5, 51.0, 50.8,50.77, 50.1, 49.9, 49.1, 48.4, 47.2, 28.4, 28.3, 28.29; MS (MALDI-TOF)m/z 2125 (M⁺+Na); HRMS (MALDI-TOF) calcd for C₇₉H₁₁₀F₂₇N₇NaO₂₇2124.6916, found 2124.7023.

r. Surfactant 22

The compound was prepared as shown in Schemes 3-9 using proceduresanalogous to those disclosed for lower analogues. ¹H NMR (400 MHz,CD₃OD) δ 4.39 (s, 2H), 4.35 (s, 2H), 4.21-4.24 (m, 14H), 4.18 (s, 6H),4.05-4.10 (m, 10H), 3.54-3.67 (m, 116H), 3.42-3.47 (m, 8H), 3.36-3.40(m, 8H); ¹⁹F NMR (376 MHz, CD₃OD) δ−71.11 (s); ¹³C NMR (100.7 MHz,CD₃OD) δ 172.1, 171.7, 171.6, 171.59, 171.3, 171.26, 171.1, 171.06,170.8, 170.7, 170.6, 170.5, 121.5 (q, J=292.6 Hz), 80.2-81.6 (m), 73.6,71.5, 71.3, 71.2, 71.17, 71.1, 70.35, 70.3, 70.2, 69.4, 68.6, 68.3,62.2, 53.3, 53.2, 53.1, 53.08, 52.9, 52.7, 50.5, 50.3, 49.5, 49.3, 48.1,47.2, 40.5, 40.47, 40.4; MS (MALDI-TOF) m/z 3077 (++Na, 100); HRMS(MALDI-TOF) calcd for C₁₁₁H₁₈₂F₂₇N₁₅NaO₅₁ 3077.1576, found 3077.1570.

9. Synthesis of Highly Fluorinated Chelators

a. Triol 3

To a stirred suspension of pentaerythritol 2 (68.0 g, 0.5 mol) intoluene (50.0 mL) at rt. was added triethyl orthoacetate (81.0 g, 92.0mL, 0.5 mol) and p-toluenesulfonic acid monohydrate (0.3 g). Thenethanol was removed by distillation from the mixture at 80° C.overnight. After all the ethanol had been distilled, the bathtemperature was raised to 125° C., and toluene was distilled off untilthe solution became homogeneous. The residue was purified by columnchromatography on neutral aluminum oxide (n-Hexane/Ethyl acetate=1/1) togive the alcohol intermediate as white solid (73.6 g, 92% yield). ¹H NMR(400 MHz, CDCl₃) δ 4.00 (s, 6H), 3.44 (s, 2H), 1.44 (s, 3H). Powderedpotassium hydroxide (123.2 g, 2.2 mol) was added to a stirred dimethylsulfoxide (750 mL), and the resulting mixture was stirred at rt. for 10min. Then the alcohol intermediate (73.6 g, 460.0 mol) was added,followed quickly by benzyl bromide (94.7 g, 65.9 mL, 554.0 mmol). Thereaction mixture was stirred for 2 h, then diluted with water (3000 mL)and extracted with diethyl ether. The combined organic phases werewashed with brine and water, dried with magnesium sulfate andconcentrated to afford the4-benzyloxymethyl-1-methyl-2,6,7-trioxa-bicyclo[2.2.2]octaneintermediate as a white solid. The intermediate was then dissolved inmethanol (300 mL) and treated with 0.1N HCl (600 mL). The resultingmixture was stirred at rt. for 4 h, treated with sodium bicarbonate(42.5 g, 506.0 mmol), stirred for an additional 1 h, and concentratedunder vacuum. The residue was purified by flash chromatography on silicagel (CH₂Cl₂/Methanol=10/1) to give the pure triol 3 as viscous oil (59.3g, 57% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.26-7.33 (m, 5H), 4.46 (s,2H), 3.64 (s, 6H), 3.39 (s, 2H).

b. Perfluoro-Tert-Butyl Ether 4

To a stirred suspension of triol 3 (11.30 g, 50.0 mmol),triphenylphosphine (59.0 g, 225.1 mmol), and 4 Å molecular sieves (6.0g) in tetrahydrofuran (300 mL) at 0° C. was added dropwisediethylazodicarboxylate (39.2 g, 225.1 mmol). After the addition, thereaction mixture was allowed to warm to rt. and stirred for anadditional 20 min. Then perfluoro-tert-butanol (53.2 g, 225.1 mmol) wasadded in one portion, and the resulting mixture was stirred for 30 h at45° C. in a sealed vessel. Water (30 mL) was added to the reactionmixture and stirred for an additional 10 min. Then the mixture wastransferred to a separatory funnel, and the lower phase was collected.Removal of low boiling point impurities under vacuum gave the product 4as clear oil (35.6 g, 81% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.25-7.35(m, 5H), 4.47 (s, 2H), 4.08 (s, 6H), 3.45 (s, 2H).

c. Alcohol 5

To a stirred solution of ether 4 (29.3 g, 33.2 mmol) and anisole (14.4g, 132.9 mmol) in dichloromethane (500 mL) at 0° C. was added aluminumchloride powder (13.3 g, 99.7 mmol) slowly. The resulting mixture wasstirred at 0° C. for 1 h, and then water (100 mL) was added slowly. Thelower layer was collected as clear oil of alcohol 5 (25.9 g, 99% yield).¹H NMR (400 MHz, Acetone-d6) δ 4.27 (s, 6H), 3.74 (s, 2).

d. Trifluoromethanesulfonate 36

To a stirred solution of alcohol 5 (7.9 g, 10.0 mmol) and pyridine (8.2mL, 7.9 g, 100.0 mmol) in tetrahydrofuran (250 mL) was added dropwise asolution of trifluoromethanesulfonic anhydride (8.2 mL, 14.1 g, 50.0mmol) in tetrahydrofuran (20 mL) at 0° C. After stirring at thistemperature for 1 h, the reaction was quenched by slow addition of water(27 mL). The mixture was transferred to separatory funnel, and the lowerphase was collected as clear oil. Washing the oil with dichloromethanegave the pure trifluoromethanesulfonate 36 as clear oil (8.8 g, 96%yield). Preferably, this compound is handled and stored at temperaturesbelow room temperature. ¹H NMR (400 MHz, Acetone-d6) δ 4.82 (s, 2H),4.39 (s, 6H); ¹⁹F NMR (376 MHz, Acetone-d6) δ 71.21 (s, 27F), 75.93 (s,3F).

e. Compound 37

A suspension of trifluoromethanesulfonate 36 (8.5 g, 9.3 mmol) andcyclen (3.3 g, 18.9 mmol) in a mixture of tetrahydrofuran anddimethylformamide (50 mL/50 mL) was stirred at rt. for 2 h. Then thereaction temperature was slowly raised to 60° C., and the mixture wasthen stirred at this temperature overnight. After concentration of thereaction mixture to dryness, the residue was dissolved indichloromethane (50 mL), and the solution was extracted with F362 (50mL, three times). Evaporation of the combined F362 phase gave theproduct 37 as clear oil (7.7 g, 88% yield). ¹H NMR (400 MHz, CD₃Cl₃) δ4.19 (s, 6H), 2.72-2.75 (m, 6H), 2.58-2.60 (m, 4H), 2.51-2.54 (m, 8H);¹⁹F NMR (376 MHz, CD₃Cl₃) δ−73.31 (s); ¹³C NMR (100.7 MHz, CD₃Cl₃) δ120.1 (q, J=293.3 Hz), 79.1-80.0 (m), 68.4, 54.4, 53.4, 46.9, 46.0,45.7, 45.5; MS (CI) m/z 945 (M⁺+1, 100); HRMS (CI) calcd forC₂₅H₂₈F₂₇N₄O₃ 945.1730, found 945.1717.

f. Compound 38

Powdered potassium carbonate (8.3 g, 60.2 mmol) and ethyl bromoacetate(4.2 mL, 6.3 g, 37.5 mmol) was added to a stirred solution of amine 37(7.1 g, 7.5 mmol) in a mixture of tetrahydrofuran and dimethylformamide(35 mL/35 mL) at rt. and the resulting suspension was stirred at 60° C.overnight. After filtration, the solvent was removed under vacuum, andthe residue was purified by flash chromatography on neutral aluminumoxide (CH₂Cl₂/Methanol=10/1) to give the product 38 as clear oil (6.3 g,92% yield). ¹H NMR (400 MHz, CD₃Cl₃) δ 4.03-4.10 (m, 12H), 3.34 (s, 2H),3.25 (s, 4H), 2.62-2.76 (m, 18H), 1.16-1.21 (m, 9H); ¹⁹F NMR (376 MHz,CD₃Cl₃) δ−73.42 (s); ¹³C NMR (100.7 MHz, CD₃Cl₃) δ 171.7, 171.4, 120.3(q, J=293.3 Hz), 79.1-80.0 (m), 68.3, 60.2, 56.0, 54.9, 54.8, 54.0,52.6, 52.2, 51.9, 46.7, 29.8, 14.3, 14.1; MS (MALDI-TOF) m/z 1023 (M⁺+1,100); HRMS (MALDI-TOF) calcd for C₃₇H₄₆F₂₇N₄O₉ 1023.2834, found1023.2883.

g. Compound 35

To a solution of lithium hydroxide (1.4 g, 60.0 mmol) in water (10 mL)was added a solution of ester 38 (6.1 g, 6.0 mmol) in methanol (200 mL)at rt. The resulted mixture was stirred at rt. overnight. Then 2N HClwas added to adjust the reaction mixture to pH 1. The solid wascollected and washed with water and diethyl ether. Solvent was thenremoved under vacuum to give the product as white solid (5.4 g, 97%yield). ¹H NMR (400 MHz, CD₃OD) δ 4.20 (s, 6H), 3.59 (s, 2H), 3.41 (s,3H), 3.22 (br, 4H), 3.06 (br, 4H), 2.98 (s, 8H), 2.80 (br, 3H); ¹⁹F NMR(376 MHz, CD₃OD) δ−70.93 (s); ¹³C NMR (100.7 MHz, CD₃OD) δ 180.1, 177.4,121.6 (q, J=292.5 Hz), 81.0 (m), 69.9, 58.5, 56.8, 52.2, 51.9, 48.9,45.9, 45.7, 22.2; MS (MALDI-TOF) m/z 1119 (M⁺+1, 100); HRMS (CI) calcdfor C₃₁H₃₄F₂₇N₄O₉ 1119.1895, found 945.1717.

h. Alcohol 7′

To a stirred solution of tetraethylene glycol 6 (97.0 g, 500.0 mmol) intetrahydrofuran (450 mL) at 0° C. was added sodium hydride (60% inparaffin, 20.8 g, 520.0 mmol) slowly, and the resulting mixture wasstirred at rt. for 30 min. Then benzyl bromide (51.2 g, 300.0 mmol) wasadded, and the resulting mixture was stirred at rt. overnight. Afterquenching the reaction with water (200 mL), the mixture was extractedwith ethyl acetate (100 mL, 4 times). The combined organic phase wasdried over anhydrous magnesium sulfate. After concentration undervacuum, the residue was purified by flash chromatography on silica gel(n-hexane/ethyl acetate=1/1) to give alcohol 7′ as clear oil (76.5 g,90%). ¹H NMR (400 MHz, CDCl₃) δ 7.28-7.29 (m, 5H), 4.51 (s, 2H),3.52-3.66 (m, 16H).

L. Methanesulfonate 8′

To a stirred solution of alcohol 7′ (69.3 g, 243.7 mmol) andtriethylamine (49.2 g, 68.4 mL, 487.0 mmol) in CH₂Cl₂ (700 mL) at 0° C.was added methanesulfonyl chloride (41.9 g, 28.3 mL, 365.6 mmol). Theresulting mixture was stirred at rt. overnight and quenched with water(400 mL). The organic phase was collected, and the aqueous phase wasextracted with ethyl acetate. The combined organic phase was washed with2N HCl (100 mL) and brine (100 mL) and then dried over anhydrousmagnesium sulfate. Concentration of the solution under vacuum gave themethanesulfonate 8′ as clear oil (86.6 g, 98%). ¹H NMR (400 MHz, CD₃Cl₃)δ 7.25-7.32 (m, 5H), 4.54 (s, 2H), 4.32-4.34 (m, 2H), 3.71-3.73 (m, 2H),3.59-3.66 (m, 12H), 3.03 (s, 3H).

j. Compound 9

A solution of alcohol 5 (5.9 g, 7.5 mmol) in tetrahydrofuran (50 mL) wasstirred at 0° C., and potassium hydride (25%, 1.4 g, 8.5 mmol) was addedslowly to the solution. After the addition, the mixture was stirred foradditional 10 min at 0° C. and methanesulfonate 8′ (4.2 g, 7.5 mmol) wasthen added in one portion. The resulting mixture was stirred at rt.overnight and quenched with water (100 mL). The organic phase wascollected, and the aqueous phase was extracted with ethyl acetate. Thecombined organic phase was washed with 2N HCl (100 mL) and brine (100mL) and then dried over anhydrous magnesium sulfate. Concentration undervacuum and flash chromatography on silica gel (n-hexane/ethylacetate=10/1) gave compound 9 as clear oil (6.4 g, 80%). ¹H NMR (400MHz, CD₃Cl₃) δ 7.26-7.33 (m, 5H), 4.56 (s, 2H), 4.07 (s, 6H), 3.54-3.69(m, 16H), 3.45 (s, 2H); ¹⁹F NMR (376 MHz, CD₃Cl₃) δ−73.28 (s); ¹³C NMR(100.7 MHz, CD₃Cl₃) δ 138.3, 128.3, 127.7, 127.5, 120.1 (q, J=292.6 Hz),78.9-79.8 (m), 73.2, 70.7, 70.65, 70.6, 70.56, 70.3, 69.4, 66.3, 65.5,46.2; MS (MALDI-TOF) m/z 1079 (M⁺+Na, 100); HRMS (MALDI-TOF) calcd forC₃₂H₃₁F₂₇NaO₈ 1079.1458, found 1079.1517.

k. Alcohol 10

A suspension of compound 9 (6.0 g, 5.7 mmol) and palladium hydroxide(10%, 1.2 g) in methanol (80 mL) was stirred under an atmosphere ofhydrogen for 2 h. After filtration, the mixture was concentrated undervacuum and purified by flash chromatography on silica gel(n-hexane/ethyl acetate=8/1) to give alcohol 10 as clear oil (5.3 g,97%). ¹H NMR (400 MHz, CDCl₃) δ 3.99 (s, 6H), 3.53-3.60 (m, 16H), 3.39(s, 2H).

1. Methanesulfonate 11

To a stirred solution of alcohol 10 (5.1 g, 5.3 mmol) and triethylamine(3.2 g, 31.8 mmol) in CH₂Cl₂ (80 mL) at 0° C. was added methanesulfonylchloride (1.9 g, 15.9 mmol). The resulting mixture was stirred at rt.and quenched with water (50 mL). The organic phase was collected, andthe aqueous phase was extracted with ethyl acetate. The combined organicsolvent was dried over anhydrous magnesium sulfate. After concentrationunder vacuum, the residue was purified by flash chromatography on silicagel (n-hexane/ethyl acetate=10/1) to give methanesulfonate 11 as clearoil (5.5 g, 99%). ¹H NMR (400 MHz, CD₃Cl₃) δ 4.35 (m, 2H), 4.04 (s, 6H),3.73-3.76 (m, 2H), 3.53-3.67 (m, 12H), 3.43 (s, 2H), 3.04 (s, 3H).

m. Compound 12

A suspension of methanesulfonate 11 (5.3 g, 5.1 mmol) and cyclen (1.8 g,10.2 mmol) in a mixture of dimethylformamide and tetrahydrofuran (50 mL)was stirred at 60° C. overnight. After concentrating the reactionmixture to dryness, the residue was purified by solid phase extractionon fluorous silica gel to give the product 12 as clear oil (5.2 g, 93%yield). ¹H NMR (400 MHz, CD₃OD) δ 4.14 (s, 6H), 3.57-3.69 (m, 14H), 3.49(s, 2H), 2.95-3.04 (m, 7H), 2.85-2.89 (m, 5H), 2.76-2.82 (m, 2H), 2.69(s, 4H); ¹⁹F NMR (376 MHz, CD₃OD) δ−71.14 (s); ¹³C NMR (100.7 MHz,CD₃OD) δ 121.6 (q, J=292.5 Hz), 80.5-81.4 (m), 78.3, 72.0, 71.9, 71.73,71.7, 71.67, 71.6, 71.56, 71.5, 71.4, 71.41, 71.4, 71.2, 69.6, 67.5,67.2, 50.9, 46.4, 44.4; MS (MALDI-TOF) m/z 1121 (M⁺+1, 100); HRMS(MALDI-TOF) calcd for C₃₃H₄₄F₂₇N₇O₄ 1021.2779, found 1021.2790.

n. Tri-Ethyl Ester 50

To a stirred solution of compound 12 (5.1 g, 4.6 mmol) intetrahydrofuran (25 mL) and dimethylformamide (25 mL) was added powderedanhydrous potassium carbonate (6.3 g, 46.0 mmol) and ethyl bromoacetate(2.6 mL, 3.8 g, 23.0 mmol). The resulting mixture was stirred overnightat 60° C. After filtration, the solvent was removed under vacuum, andthe residue was purified by flash chromatography on neutral aluminumoxide (CH₂Cl₂/Methanol=10/1) to give the product 50 as clear oil (5.2 g,82% yield). ¹H NMR (400 MHz, CD₃OD) δ 4.16-4.29 (m, 8H), 4.14 (s, 6H),3.56-3.67 (m, 16H), 3.48 (s, 4H), 2.70-3.45 (m, 4H) 2.41-2.76 (m, 14H),1.27-1.30 (m, 9H); ¹⁹F NMR (376 MHz, CD₃OD) δ−71.12 (s); ¹³C NMR (100.7MHz, CD₃OD) δ 175.3, 175.1, 121.6 (q, J=292.6 Hz), 80.7-81.5 (m), 71.9,71.5, 71.4, 71.3, 71.2, 70.8, 68.5, 67.4, 67.1, 62.3, 62.26, 56.4, 56.1,53.6, 51.9, 51.1, 48.4, 47.4, 14.5, 14.3; MS (MALDI-TOF) m/z 1379 (M+1,100); HRMS (MALDI-TOF) calcd for C₄₅H₆₂F₂₇N₄O₁₃ 1379.3882, found1379.3902.

o. Compound 1

Lithium hydroxide (0.9 g, 37.0 mmol) was added to a solution of compound9 (5.1 g, 3.7 mmol) in methanol (100 mL) and water (10 mL). Theresulting mixture was stirred at rt. for 8 h. Then 1N HCl was added toadjust the solution to pH 1. After removal of solvent under vacuum, theresidue was purified by flash column chromatography on neutral aluminumoxide to give compound 1 as white solid (4.7 g, 990%). ¹H NMR (400 MHz,Acetone-d6) δ 4.20 (s, 6H), 3.53-3.59 (m, 16H), 2.2-3.2 (m, 24H); ¹⁹FNMR (376 MHz, CD₃OD) δ−71.15 (s); ¹³C NMR (100.7 MHz, CD₃OD) δ 180.9,179.6, 175.3, 175.1, 121.5 (q, J=292.6 Hz), 80.5-81.4 (m), 71.9, 71.6,71.5, 71.4, 71.3, 69.9, 68.8, 67.5, 67.1, 60.6, 59.2, 53.9, 52.4, 51.9,47.4; MS (MALDI-TOF) m/z 1295 (M⁺+1, 100); HRMS (MALDI-TOF) calcd forC₃₉H₅₀F₂₇N₄O₁₃ 1295.2943, found 1295.2953.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A compound comprising the structure:

wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, and R₃₃ are CF₃; andwherein R₄ is H, OH, OBn, OC(CF₃)₃, alkyl, or alkoxy.
 2. The compound ofclaim 1, wherein the compound is a surfactant.
 3. The compound of claim1, comprising a structure selected from:

wherein n is from 2 to
 10. 4. The compound of claim 1, wherein thecompound is an oil.
 5. The compound of claim 1, comprising a structureselected from:


6. The compound of claim 1, wherein the compound exhibits maximumsymmetric branching.
 7. A process for the preparation of a compoundcomprising the structure:

wherein R is CF₃ and wherein R₄ is H, OH, OBn, alkyl, or alkoxy; theprocess comprising the steps of: providing a triol, reacting the triolwith or nonafluoro-tert-butanol to provide a triperfluoro-tert-butylether.
 8. The process of claim 7, wherein the compound comprises thestructure:

wherein n is 0 or a positive integer; wherein R₅₁, R₅₂, R₆₁, and R₆₂are, independently, H or alkyl; and wherein R₇, R₈, and R₉ are,independently, H, CH₂CO₂H, or alkyl.
 9. The process of claim 8, whereinn is an integer from 4 to
 12. 10. A delivery method comprising the stepsof: a. complexing a payload with one or more compounds of claim 6; andb. administering the complex to a mammal in an effective amount.
 11. Themethod of claim 10, comprising the steps of: a. complexing a metal ionwith a compound of claim 6; and b. administering the complex to asubject in an amount effective for detection by ¹H MRI.
 12. The methodof claim 10, comprising the steps of: a. complexing a radionuclide witha compound of claim 6; and b. administering the complex to a mammal inan amount effective for radiotherapy.